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Molecular Biology of the Cell • 35:ar40, 1–24, March 1, 2024 35:ar40, 1
M BoC | ARTICLE
Loss of the HOPS complex disrupts early-to-late
endosome transition, impairs endosomal
recycling and induces accumulation of
amphisomes
ABSTRACT The multisubunit HOPS tethering complex is a well-established regulator of lyso-
some fusion with late endosomes and autophagosomes. However, the role of the HOPS
complex in other stages of endo-lysosomal trafficking is not well understood. To address this,
we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the
HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endo-
cytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area.
By correlative light-electron microscopy, these endosomes showed a complex ultrastructure
and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as
well as late (Rab7, CD63, LAMP1) endosomes. These “HOPS bodies” were not acidified, con-
tained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-in-
dependent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted
from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced.
Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them
as amphisomes. Together, these findings show that depletion of the lysosomal HOPS com-
plex has a profound impact on the functional organization of the entire endosomal system
and suggest the existence of a HOPS-independent mechanism for amphisome formation.
Monitoring Editor
Sharon Tooze
The Francis Crick Institute
Received: Aug 29, 2023
Revised: Dec 22, 2023
Accepted: Jan 5, 2024
© 2024 van der Beek etal. This article is distributed by The American Society for
Cell Biology under license from the author(s). It is available to the public under an
Attribution 4.0 International Creative Commons CC-BY 4.0 License (https://
creativecommons.org/licenses/by/4.0/).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society for Cell Biology.
IF, immunofluorescence; ILVs, intraluminal vesicles; KO, knock-out; LAMP1, lyso-
some-associated membrane protein 1; LC3, microtubule-associated proteins
1A/1B light chain 3B; M6P, mannose-6-phosphate; PtdIns3P, phosphatidylinositol
3-phosphate; SNARE, soluble N-ethylmaleimide-sensitive factor attachment pro-
tein receptor; SNX1, sorting nexin 1; TEM, transmission electron microscope;
TGN, trans-golgi network; Vps, vacuolar protein sorting; WB, western blot.
Jan van der Beek, Cecilia de Heus, Paolo Sanza, Nalan Liv, and Judith Klumperman*
Center for Molecular Medicine, University Medical Center Utrecht, Institute of Biomembranes, Utrecht University,
3584 CX Utrecht, The Netherlands
SIGNIFICANCE STATEMENT
• The HOPS tethering complex is a well-established regulator of lysosomal fusion. Additionally, it has
proposed, but unclear roles in other pathways like endosomal maturation. By knock-out of HOPS
subunits, we nd an overall disorganization of the endosomal compartment..
• Specically, we nd enlarged compartments with hybrid early (Rab5, EEA1, PI3P) and late (Rab7,
cathepsin D, LAMP1) endosomal markers, which we call HOPS bodies. HOPS bodies also fail to
recycle CI-MPR, are not acidied (pH 5.9) and contain autophagic cargo.
• Our results suggest a broader role for HOPS in endo-lysosomal organization and a HOPS-indepen-
dent mechanism by which autophagosomes fuse with endosomes.
Special Issue on Cell Biology of the Lysosome
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E23-08-0328) on January 10, 2024.
Conflicts of interest: The authors declare no financial conflicts of interests.
Author contributions: J.B., P.S., N.L., and J.K. concieved and designed the
experiments; J.B., C.D., and P.S. performed the experiments; J.B. analyzed the
data; J.B. and J.K. drafted the Article; J.B. prepared the digital images.
*Address correspondence to: Judith Klumperman (J.Klumperman@umcutrecht.nl).
Abbreviations used: CatD, cathepsin D; CI-MPR, cation-independent mannose-
6-phosphate receptor; CLEM, correlative light-electron microscopy; CORVET,
class C core vacuole/endosome tethering; EEA1, early endosomal antigen 1; EM,
electron microscopy; ESCRT, endosomal sorting complexes required for transport;
HOPS, homotypic fusion and vacuole protein sorting; HPF, high-pressure freezing;
2 | J. van der Beek et al. Molecular Biology of the Cell
INTRODUCTION
Membrane-bound organelles are a dening trait of eukaryotic cells.
The endo-lysosomal system regulates the turnover of external and
internal molecules and plays a key role in nutrient sensing and cel-
lular homeostasis (Ballabio and Bonifacino, 2020). Disruption of the
endo-lysosomal system underlies many genetic and acquired dis-
eases, including lysosomal storage disorders, neurodegenerative
diseases, and cancer (Hämälistö and Jäättelä, 2016; Lie and Nixon,
2019; Marques and Saftig, 2019).
To carry out its various tasks, the endo-lysosomal system is orga-
nized as an intricate network of distinct compartments with special-
ized functions. Small endocytic vesicles form by budding from the
plasma membrane and fuse with early endosomes, which sort cargo
for recycling back to the plasma membrane or degradation. Early
endosomes mature into late endosomes (Saftig and Klumperman,
2009; Huotari and Helenius, 2011), which package membrane pro-
teins destined for degradation into intraluminal vesicles (ILVs), or
sort proteins for retrograde transport to the trans-Golgi network
(TGN). Late endosomes fuse with lysosomes containing active hy-
drolases, after which cargo is degraded and surplus membranes
retrieved for endo-lysosomal reformation (Yang and Wang, 2021). In
addition, cells target intracellular cytoplasmic cargo like proteins,
aggregates, and organelles for degradation by incorporation into
autophagosomes (Hu and Reggiori, 2022). The forming, double
membranous autophagosomes select cytoplasmic cargoes through
interaction of autophagy adaptors as p62/SQSTM1 with Atg8-family
proteins, such as LC3. Autophagosomes fuse with late endosomes
or lysosomes, forming amphisomes or autolysosomes respectively,
both eventually resulting in lysosomal degradation of the autopha-
gic cargo (Lőrincz and Juhász, 2020). Finally, endo-lysosomal organ-
elles play a key role in various signaling pathways (Ballabio and
Bonifacino, 2020), cell-type−specic transport steps (Delevoye
etal., 2019) and processes such as cell division and migration (Pu
etal., 2016; Hämälistö etal., 2020). As a whole, the endo-lysosomal
system is a key regulatory system for cellular metabolism and
homeostasis.
The formation of the distinct endo-lysosomal compartments re-
quires several membrane fusion steps, which are orchestrated by
specic combinations of Rab GTPases, SNAREs, and tethers. Rab
GTPases are central regulators of membrane trafcking (Stenmark,
2009). After switching from a GDP-bound inactive state to a GTP-
bound active state, which is promoted by Guanine Exchange Factors
(GEFs), activated Rabs associate with explicit membranes and recruit
various effector proteins that control protein sorting, membrane
composition, organelle positioning, and fusion. Rab5 is the dening
GTPase on endocytic vesicles and early endosomes, and is replaced
by Rab7 on late endosomes and lysosomes (Rink etal., 2005). The
switch from Rab5 to Rab7 occurs during early-to-late endosomal
maturation and is mediated by the Mon1−Ccz1 complex, which acts
as an inhibitor of Rab5 and GEF for Rab7 (Poteryaev et al., 2010;
Huotari and Helenius, 2011; Langemeyer et al., 2020; van den
Boomen et al., 2020; Herrmann et al., 2023). To enact early endo-
some fusion events, Rab5 recruits the multisubunit tethering com-
plex (MTC) “class C core vacuole-endosome tethering” (CORVET)
(Peplowska etal., 2007; Perini etal., 2014; Gillingham etal., 2019).
Similarly, on late endosomes Rab7, possibly via or in addition to
Rab2, recruits the related “homotypic fusion and vacuole protein
sorting” (HOPS) complex that governs lysosomal fusion events
(Rieder and Emr, 1997; Seals et al., 2000; Lin et al., 2014; Lőrincz
etal., 2017; Jongsma etal., 2020; Schleinitz etal., 2023;).
The HOPS complex is well characterized in yeast and mamma-
lian cells (Seals etal., 2000; Wurmser etal., 2000; Balderhaar and
Ungermann, 2013; Van Der Kant et al., 2015;), with extensive in
vitro work on its structure (Brocker etal., 2012; Chou etal., 2016;
Shvarev et al., 2022) and fusogenic activity (Zick and Wickner,
2013; Schwartz etal., 2017; Song etal., 2020). The HOPS complex
consists of six subunits; the core units Vps11, Vps18, Vps16, and
Vps33A that are shared with CORVET, and Vps39 and Vps41 that
are HOPS-specic (Seals etal., 2000; Van Der Kant etal., 2015; van
der Beek etal., 2019; Shvarev et al., 2022) (Figure 1A). Multiple
HOPS subunits interact with Rab7 and its effectors RILP and
PLEKHM1 (McEwan et al., 2015; Van Der Kant et al., 2015;
Jongsma etal., 2020; Zhang etal., 2023). Additionally, Vps39 in-
teracts with Rab2 (Kajiho etal., 2016; Schleinitz et al., 2023) and
Vps41 with the lysosomal GTPase Arl8b (Khatter etal., 2015). By
acting as a tether between these GTPases, HOPS brings specic
membranes in close proximity (Shvarev etal., 2022). Furthermore,
the core components Vps33A and Vps16 promote assembly of
soluble N-ethylmaleimide-sensitive fusion protein (NSF) attach-
ment protein receptor (SNARE) complexes (Graham etal., 2013;
Baker et al., 2015; D’Agostino et al., 2017; Song et al., 2020).
Complex formation of cognate SNAREs on opposing membranes
drives membrane fusion. Both endo-lysosomal (VAMP7, VAMP8,
Vti1b, Stx7, Stx8) (Sato etal., 2000; Kim etal., 2001; Collins etal.,
2005; Stroupe etal., 2006; Collins and Wickner, 2007; Luzio etal.,
2009) and autophagosomal (Stx17, SNAP29) (Jiang et al., 2014;
Takáts et al., 2014) SNAREs interact with the HOPS complex.
Hence, through various interactions, the HOPS complex promotes
multiple fusion events between lysosomes, late endosomes, and
autophagosomes (Pols etal., 2013a; Jiang etal., 2014; Wartosch
etal., 2015).
An increasing number of studies report mutations in HOPS sub-
units as the cause of rare neurological disorders, often associated
with dystonia (Steel etal., 2020; Monfrini et al., 2021a; Monfrini
etal., 2021b; van der Welle etal., 2021; van der Beek etal., 2019).
On a cellular level, depletion or disruption of the HOPS complex
invariably impairs endocytic progression to lysosomes and de-
creases autophagic ux (Pols et al., 2013a; Jiang et al., 2014;
Wartosch etal., 2015). We previously also found that small inter-
fering RNA (siRNA)-mediated knockdown of Vps41 or Vps39 re-
sulted in an accumulation of late endosomes (Pols etal., 2013a).
Additionally, depletion of HOPS components leads to phenotypes
that are not straightforwardly explained by a defect in endo-lyso-
somal fusion. In Drosophila, Vps18 (dor) and Vps33A (car) mutant
ies are defective in delivery of biosynthetic cathepsin D to endo-
somes (Sriram etal., 2003) and Vps18 conditional knock-out (KO)
mice accumulate the proform of cathepsin D in affected tissues
(Peng et al., 2012). However, Vps41 patient cells or HOPS-de-
pleted cells still contain lysosomes with active cathepsins (Wartosch
etal., 2015; van der Welle etal., 2021). Furthermore, the HOPS
complex has been implicated in Rab GTPase switching on endo-
somes and lysosomes. Loss of Vps39 delays the Rab5 to Rab7
switch (Rink et al., 2005) and the HOPS complex interacts with
Mon1−Ccz1 (Poteryaev etal., 2010). It also cooperates with SKIP,
an Arl8b effector, for switch of Rab7 to Arl8b (Jongsma et al.,
2020). However, the precise role of the HOPS complex in Rab con-
version remains elusive.
The better understand the role of the HOPS complex in vesicular
trafcking pathways other than lysosomal fusion, we here investi-
gated the effect of HOPS depletion on early−late endosome forma-
tion and function. We nd that deletion of distinct HOPS subunits
invariably results in the accumulation of enlarged endosomes with
mixed early and late characteristics. These “HOPS bodies” are
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 3
enzymatically inactive, fail to recycle the cation-independent man-
nose-6-phosphate receptor (CI-MPR) and accumulate endocytic and
autophagic cargo. Overall, our data show that loss of the HOPS
complex leads to a general disruption of endosomal identity and
function, highlighting a central role for HOPS in endo-lysosomal or-
ganization. Strikingly, our data also indicate that endosomes can
incorporate macroautophagic material in a HOPS-independent
manner.
FIGURE 1: Depletion of HOPS increases Rab5 and Rab7 colocalization. (A) Schematic representation of the HOPS
complex structure based on yeast (Shvarev etal., 2022). (B) Western blot of HeLa cell lines knocked-out (KO) for HOPS
subunits Vps11, Vps18, Vps39 or Vps41 by CRISPR/Cas9. KO of one subunit generally leads to a decrease in others.
Vps39 KO was confirmed using DNA sequencing (van der Welle etal., 2021). (C) HeLa WT and KO cells incubated with
SiRlysosome, which marks active cathepsin D, and Dextran-AF488 for 2 h, fixed, and analyzed by widefield microscopy.
(D) Quantification of the percentage of active cathepsin D compartments reached by endocytosed Dextran-AF488 after
2-h uptake. All HOPS Kos showed reduced colocalization, indicating that active lysosomes are poorly reached by
endocytosed material, a key readout for HOPS inactivation. N ≥ 55 for each condition. (E) IF of Rab5 and Rab7
Vps11 KO cells. Enlarged Rab5 endosomes are also positive for Rab7 (arrows). (F) Quantification of Rab5-positive
compartments colocalizing with Rab7 in WT cells and after KO of indicated HOPS subunits (representative images
shown in Fig. S1A). Rab5 and Rab7 colocalization increases upon HOPS subunit depletion. n = 45, 32, 21, 49 and 17 for
WT, 11KO, 18KO, 39KO, and 41KO from two independent replicates, respectively. Changes from WT are significant at
p < 0.01 (**) or p < 0.001 (***), by Kruskal–Wallis multiple comparison test. Dots in dot plots represent individual cells.
Scale bars 20 µm in overview images, 5 µm in insets.
4 | J. van der Beek et al. Molecular Biology of the Cell
RESULTS
Knockout of individual HOPS subunits invariably impairs
endocytic traffic to active lysosomes
To investigate the role of the HOPS complex on the endo-lysosomal
system, we generated knockout (KO) HeLa cell lines of Vps11,
Vps18, Vps39, and Vps41 using CRISPR/Cas9 (van der Welle etal.,
2021). This selection encompassed core subunits forming the
backbone of the CORVET/HOPS complex (Vps11, Vps18), as well as
the HOPS-specic subunits Vps39 and Vps41 (Figure 1A). We as-
sessed the loss of Vps11, Vps18, and Vps41 by Western blot analy-
sis (Figure 1B) and, due to a lack of suitable antibodies, of Vps39
through genomic sequencing (van der Welle etal., 2021). The West-
ern blots conrmed deletion of Vps18 and Vps41 and showed an
almost complete removal of Vps11 (>95%) in the respective KO
lines. The phenotypical similarity of the Vps11 KO cell line to other
HOPS KO lines in all subsequent assays indicated that the remaining
band is either background, nonfunctional, or too little protein to
establish fusogenic activity. The Western blot additionally revealed
that upon KO of one subunit, others were also partially depleted,
suggesting that subunit stability in part relies on their incorporation
into the HOPS complex.
Depletion of the HOPS complex impairs delivery of endocytic
cargo to active lysosomes (Pols etal., 2013a; Wartosch etal., 2015).
Therefore, to functionally validate the different HOPS KO cell lines,
we assessed in each cell line the delivery of endocytosed (2 h) Dex-
tran conjugated to AlexaFluor (AF)-488 to enzymatically active lyso-
somes marked with SiRlysosome (Figure 1C) (Wartosch etal., 2015;
van der Welle etal., 2021). Dextran-AF488 uorescence is visible in
each compartment reached by endocytosis. SiRlysosome only binds
to cathepsin D in its active form, which is restricted to the acidied
milieu of the lysosome. In agreement with previous studies, the
percentage of SiRlysosome-positive compartments reached by
Dextran-AF488 was signicantly reduced in all 4 HOPS KO cells
(Figure 1D). This indicated that all HOPS KO cell lines were defective
in the delivery of endocytosed cargo to active lysosomes. Moreover,
the data showed that the HOPS KO phenotype was not overcome
by compensatory mechanisms.
HOPS subunit depletion impairs early to late endosome
maturation resulting in enlarged hybrid endosomes that
cluster in the perinuclear area
Previous studies have shown that HOPS depletion delays conver-
sion of Rab5 to Rab7, suggesting a role for HOPS in the maturation
of early to late endosomes (Rink etal., 2005). To assess Rab conver-
sion in our HOPS KO cell lines, we stained for endogenous Rab5
and Rab7 by immunouorescence (IF) and compared HeLa wild
type (WT) to Vps11, Vps18, Vps39, or Vps41 KO cells. In HeLa WT
cells, 25−30% of Rab5-positive endosomes was also positive for
Rab7. This overlap increased to ∼50% in all HOPS KO lines, in agree-
ment with a delay in Rab5 to Rab7 conversion (Figure 1E and F;
Supplementary Figure S1A). In addition, we found that hybrid Rab5-
Rab7 endosomes were enlarged and clustered in the perinuclear
area (Figure 1E, arrows; Supplementary Figure 2, A and B). Of note,
both these phenomena were reminiscent of the phenotype seen
upon expression of a constitutively active form of Rab5 (Rab5QL)
(Wegener etal., 2010), albeit less pronounced.
EEA1 is a tethering factor on early endosomes and effector of
Rab5 (Mu etal., 1995; Murray etal., 2016). By double IF labeling of
EEA1 and Rab5, we found that in HOPS KO cells the majority of
EEA1 redistributed to the perinuclear, enlarged endosomes
(Figure 2A). This redistribution was also illustrated by an overall in-
crease in size of EEA1 compartments in HOPS KO versus WT cells
(Figure 2B; Supplementary Figure S1B). We previously reported that
EEA1 marks Rab5 and Phosphatidylinositol 3-phosphate (PtdIns3P)-
positive endosomes, but is absent from Rab7-positive endosomes
(van der Beek etal., 2022). Here, in HOPS KO cells, we found that
the enlarged, perinuclear Rab5 endosomes were also positive for
Rab7 (Figure 1E and F; Supplementary Figure S1A). Double labeling
showed that in HOPS KO cells EEA1−Rab7 colocalization was sig-
nicantly increased (18−32% in KO cells) compared with WT cells
(10%; Supplementary Figure S1, C and D), with both proteins
strongly enriched on the enlarged, perinuclear endosomes. Because
EEA1 IF distinctively highlighted the population of hybrid Rab5-
Rab7−positive endosomes, we used EEA1 as marker for these or-
ganelles in our subsequent microscopy studies.
PtdIns3P is an interactor of EEA1 and important for endosomal
maturation (Huotari and Helenius, 2011). Recently, we showed by
correlative light-electron microscopy (CLEM) the existence of a sub-
population of EEA1 and PtdIns3P-positive endosomes that by mor-
phology was classied as late endosomal (van der Beek etal., 2022).
To investigate whether the enlarged, perinuclear Rab5-Rab7-EEA1
endosomes in HOPS KO cells also contained PtdIns3P, we trans-
fected these with a 2xFYVE-mCherry construct, a genetic reporter
that specically binds PtdIns3P (Gillooly etal., 2003) (Figure 2C). By
uorescence microscopy, we indeed found PtdIns3P on the typical,
EEA1-positive enlarged endosomes in HOPS KO cells. To establish
whether these Rab5-Rab7-EEA1-PtdIns3P endosomes still formed
recycling tubules, we performed IF of Vps35, a component of the
Retromer complex. Retromer controls the recycling of a variety of
cargos to either the TGN or plasma membrane (Burd and Cullen,
2014). Both in WT and Vps11 KO cells, we readily found Vps35-
positive structures associated with EEA1-positive endosomes (Sup-
plementary Figure S2A), suggesting that the hybrid endosomes
were still capable of forming recycling tubules. To further assess the
recycling capacity in HOPS KO cells, we performed a pulse chase
experiment with Transferrin-AlexaFluor488 (Tf-488), a general
marker for endosome to plasma membrane recycling (Supplemen-
tary Figure S2, B and C). After 5-min uptake, in both WT and HOPS
KO cells Tf-488 was found in EEA1-positive compartments (at
30−50% colocalization). We then chased with normal medium for
10 or 30 min, which is known to deplete cells from Tf-488 by recy-
cling and release into the medium. Correspondingly, a 10-min chase
in HeLa WT cells decreased the colocalization of Tf-488 with EEA1
to only 7% and after 30 min Tf-488 was barely detectable (colocal-
ization with EEA1 <1%). Strikingly, the HOPS KO cells showed a very
similar pattern, with after 10-min chase only 3−12% colocalization
and less than 1% colocalization after 30-min chase. These results
indicated that recycling from endosomes to the plasma membrane
remained unaffected in HOPS KO cells.
Together, these results showed that HOPS KO induced the ac-
cumulation of enlarged EEA1-Rab5-PtdIns3P-Rab7-Vps35−positive
endosomes in the perinuclear area. The hybrid early−late molecular
composition of these endosomes suggested a block or delay in the
Rab5-Rab7 switch. The data also showed that HOPS was not re-
quired to establish PtdIns3P levels on endosomes or to recycle Tf to
the plasma membrane.
HOPS KO−induced hybrid endosomes contain inactive
lysosomal enzymes
Whereas the role of HOPS complex in lysosomal fusion is well es-
tablished, studies on the role of HOPS in the delivery of lysosomal
enzymes are conicting (Sriram etal., 2003; Swetha et al., 2011;
Peng etal., 2012). To investigate a putative effect of HOPS KO on
lysosomal enzyme transport, we used a pan-cathepsin D antibody
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 5
to label both active and inactive forms of the enzyme. In HeLa WT
cells, the percentage of EEA1 compartments that contained ca-
thepsin D was only ∼12%, reecting its predominantly late endo-
lysosomal steady-state distribution. In contrast, in HOPS KO cells
cathepsin D was found in 25−40% of the EEA1-positive hybrid com-
partments (Figure 2D and E; Supplementary Figure S3A), indicating
that HOPS KO caused the accumulation of lysosomal enzymes in
these enlarged endosomes. Moreover, labeling for CD63 and
LAMP1 also showed a signicant overlap with EEA1, indicating that
the enlarged endosomes also contained late endosomal−lysosomal
membrane proteins (Supplementary Figure S3, B−E). Because wide-
eld microscopy has a limited resolution especially in the z-axis, we
then performed uorescence labeling of 150 nm−thick thawed
cryosections. With the improved z-resolution, we found striking
examples of colocalization for EEA1, LAMP1, and cathepsin D in
HOPS KO cells (Supplementary Figure S3, F and G). Together, these
data unequivocally showed that the enlarged endosomes induced
by HOPS KO contained a mixed set of early endosomal proteins,
late endo-lysosomal regulators, lysosomal enzymes, and membrane
proteins.
FIGURE 2: HOPS KO cells accumulate enlarged endosomal compartments with mixed early and late endosomal
proteins. (A) Enlarged, perinuclear Rab5-positive compartments in HOPS KO cells specifically accumulate EEA1 label.
(B) Quantification of EEA1 compartment size. Based on thresholded pixel counts in confocal images (shown in
Supplementary Figure S1B), n ≥ 27 from two independent replicates for each condition. (C) IF staining for EEA1 in cells
transfected with the 2xFYVE-mCherry probe for PtdIns3P. Strong colocalization is observed in both HeLa WT and Vps11
KO cells. (D) IF staining for cathepsin D and EEA1 reveals striking colocalization in Vps11 KO cells, whereas
colocalization in HeLa WT cells is minimal. (E) Quantification of the percentage of EEA1-positive compartments
colocalized with cathepsin D, including other KOs (IF examples shown in Fig. S3A). n ≥ 41 from two independent
replicates for each condition. Changes from WT are significant at p < 0.001 (***), by Kruskal−Wallis multiple comparison
test. Dots in dot plots represent cell averages (B) or individual cell values (E). Scale bars 20 µm in overview images, 5 µm
in insets.
6 | J. van der Beek et al. Molecular Biology of the Cell
FIGURE 3: HOPS KO impairs cathepsin B and D processing to their active forms. (A) Western blot of cathepsin B and
cathepsin D showing relative enrichment of pro-forms in HOPS KO cells. (B) Quantification of the ratio active over
inactive cathepsin D as shown in A. Based on three independent replicates, dots represent individual replicate values.
(C) Combined staining for pan-cathepsin D and active cathepsin D using antibody and SiRlysosome, respectively.
Additional IF of EEA1 showed colocalization with pan-cathepsin D but not SiRlysosome signal. Inset is enlargement of
outlined area. (D) IF staining as in B, but with Golgi marker GM130 instead of EEA1. Pan-cathepsin D staining is
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 7
To extend these observations to other cell types, we next gener-
ated A549 cell lines KO for Vps18 and Vps41, and HT1080 cells KO
for Vps18 and Vps39. We validated these lines using Western blot
(Supplementary Figure S4, A and B) or, in the case of the HT1080
Vps39 KO, genomic sequencing (Supplementary Figure S4C; and
Materials and Methods). As a key dening experiment for the HOPS
KO phenotype found in HeLa cells, we performed Rab7-EEA1 and
cathepsin D-EEA1 colocalization by IF. Although the base level of
colocalization differed between the respective WT lines, HOPS de-
pletion invariably increased the level of colocalization between
these early and late endosomal markers (Supplementary Figure S4,
D−I). These data showed the formation of hybrid early−late endo-
somes by HOPS KO was a consistent phenotype, found in multiple
cell types.
Activation of cathepsins requires processing into their mature
forms, which generally involves an acidied environment and cleav-
age by other proteases (Laurent-Matha et al., 2006; Yadati et al.,
2020). To investigate the activity of lysosomal hydrolases in HOPS
KO cells, we analyzed the ratio of the active mature forms over the
inactive proforms by Western blot. This showed a striking decrease
for cathepsin D in HOPS KO compared with HeLa WT cells, indicat-
ing a relative enrichment in the profrom (Figure 3A and B). A less
striking but similar effect was seen for cathepsin B (Figure 3A). Next,
to determine the subcellular localization of inactive versus active
cathepsin D, we combined the pan-cathepsin D antibody with the
SiRlysosome probe in uorescent microscopy. In this setup, organ-
elles labeled for pan-cathepsin D but not SiRlysosome will only con-
tain inactive cathepsin D. In HeLa WT cells, pan-cathepsin D and
SiRlysosome signals largely overlapped and showed little colocal-
ization with EEA1, indicating that the vast majority of cathepsin D
was active and localized in late endo-lysosomal compartments
(Figure 3C). Strikingly, in HOPS KO cells the pan-cathepsin D label
colocalized less with SiRlysosome (Figure 3C; Supplementary Figure
S5A) and signicantly colocalized with the enlarged, EEA1-positive
endosomes where the SiRlysosome signal was generally absent
(Figure 3C and E). Thus, the cathepsin D present in the hybrid endo-
somes induced in HOPS KO cells was predominantly inactive.
Further examination of the pan-cathepsin D labeling in HOPS KO
cells revealed an additional pool of cathepsin D in the perinuclear
area that was not labeled for EEA1 (Figure 3C). Combining pan-
cathepsin D and SiRlysosome staining with the Golgi or TGN mark-
ers GM130 or TGN46 (Figure 3D and F; Supplementary Figure S5B),
showed a clear increase in this Golgi/TGN pool of inactive cathepsin
D in the HOPS KO cells. Together, these experiments showed that
HOPS KO cells accumulated inactive cathepsin D in the Golgi/TGN
area as well as in the hybrid early−late endosomes marked by EEA1.
The altered distribution and activity of cathepsin D in HOPS KO
cells indicated a defect in lysosomal enzyme trafcking. In case lyso-
somal enzymes lack a mannose-6-phosphate tag, they are in the
TGN incorporated in the default secretory pathway and released
into the culture medium (Reitman etal., 1981). To assess if this was
also the case in HOPS KO cells, we concentrated proteins of serum-
free culture medium from HeLa WT and HOPS KO cells by TCA
precipitation. The resulting culture medium concentrates were ana-
lyzed by Western blot. This revealed an increase in secreted pro-
forms of cathepsins B, D, and L in all HOPS KO cells (Supplementary
Figure S5C).
Combined, our data demonstrated that HOPS KO resulted in the
peri-nuclear accumulation of enlarged endosomal compartments
that acquired late endo-lysosomal proteins (LAMP1, inactive ca-
thepsin D, CD63, Rab7; Figure 2D; Supplementary Figures S1C and
S3, B and D), while early endosomal markers (Rab5, EEA1, PtdIns3P,
Vps35; Figure 2A and C; Supplementary Figures S1C and S2A) were
still present. The data imply that HOPS KO led to an impairment in
early to late endosomal maturation and decreased activation of ly-
sosomal enzymes. For clarity, we will further refer to the hybrid en-
dosomal compartments as “HOPS bodies.”
HOPS depletion impairs retrograde recycling of CI-MPR
Most lysosomal enzymes, including cathepsin D, depend on the CI-
MPR for trafcking to lysosomes. Lysosomal enzymes are tagged
with a mannose-6-phosphate (M6P) group in the Golgi complex that
is recognized by the CI-MPR in the TGN (Hille-Rehfeld, 1995;
Braulke and Bonifacino, 2009). CI-MPR and its bound ligands exit
the TGN in clathrin-coated vesicles that trafc to early endosomes
(Klumperman etal., 1993; Waguri et al., 2003). After substrate re-
lease in the acidied milieu of late endosomes, CI-MPR travels back
to the TGN by a retrograde pathway dependent on ESCPE, a com-
plex formed by SNX1/2 and 5/6 (Mari etal., 2008; Evans etal., 2020;
Simonetti etal., 2023). Our ndings that HOPS KO led to the accu-
mulation of inactive cathepsin D in the Golgi/TGN (Figure 3D and F;
and Supplementary Figure S3) raised the question whether CI-MPR
trafcking was disrupted in these cells.
To address this, we performed IF staining of CI-MPR in combina-
tion with TGN46, SNX1, and/or EEA1 in HeLa WT and HOPS KO
cells. In HeLa WT cells, the majority of CI-MPR was present in the
TGN (Figure 4A, white outlines), which is in agreement with many
previous studies (Brown and Farquhar, 1984; Brown et al., 1984;
Seaman, 2004; Styers etal., 2004). The CI-MPR signal outside the
TGN partially colocalized with EEA1 (WT in Figure 4C and D) and
more strikingly with SNX1 (WT in Figure 4A and B), indicative for the
retrograde pathway from endosomes to TGN. Strikingly, in HOPS
KO cells, the majority of CI-MPR signal localized to the EEA1-posi-
tive HOPS bodies (Figure 4C and D), outside the Golgi area
(Figure 4A and B). Since by Western blot the overall levels of CI-MPR
were not higher in the HOPS KO cells (Figure 4E), this altered
staining pattern indicated a redistribution of CI-MPR rather than in-
creased levels in endosomes due to decreased degradation. In
addition, the overall colocalization of CI-MPR with SNX1 had de-
creased in all HOPS KO cells (Figure 4A and B), suggesting a block-
ade in retrograde transport.
To reveal the association of CI-MPR with SNX1 in ultrastructural
detail, we performed immuno-electron microscopy (immuno-EM) of
WT and HOPS KO cells using immunogold labeling of cryosections
(Figure 5A–D; Supplementary Figure S6). HOPS KO cells contained
a characteristic population of enlarged endosomes with mixed early
and late morphological features which we identied as HOPS bod-
ies (see below and Figures 7 and 8). Quantitation of the relative
enriched in the Golgi area, which lacks SiRlysosome. (E) Quantification of C based on spot detection. Percentage of
EEA1-positive compartments colocalized with pan-cathepsin D without SiRlysosome signal, n ≥ 17 for each condition.
(F) Quantification as in E. The percentage of GM130 compartments positive for pan-cathepsin D but not SiRlysosome
increased in HOPS KO cells. n ≥ 22 per condition. Changes from WT are significant at p < 0.05 (*), p < 0.01 (**) or
p < 0.001 (***) or not significant (n.s.), by Kruskal−Wallis multiple comparison test. Dots in the dot plots represent
individual cells. Scale bars 20 µm in overview images, 5 µm in insets.
8 | J. van der Beek et al. Molecular Biology of the Cell
FIGURE 4: HOPS depletion impairs CI-MPR retrograde transport. (A) IF of SNX1, CI-MPR and TGN46 reveals reduced
colocalization of SNX1 and CI-MPR. Outlines in insets represent TGN46 signal. (B) Quantification of CI-MPR
colocalization with SNX1. n ≥ 48 from 2 independent replicates for each condition. (C) IF of CI-MPR and EEA1 in HeLa
WT and HOPS KO cells. CI-MPR redistributes to EEA1-positive compartments in HOPS KO cells. (D) Quantification of C
as the percentage of thresholded EEA1 signal overlapping thresholded CI-MPR signal. n ≥ 43 per condition from two
independent replicates. (E) Western blot of CI-MPR shows no significant increase in HOPS KO cells. Changes from WT
are significant at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***) by Kruskal−Wallis multiple comparison test. Dots in dot plots
represent individual cells. Scale bars 20 µm in overviews, 5 µm in insets.
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 9
distribution of CI-MPR (Figure 5E) showed that the fraction of CI-
MPR in endosomes strikingly increased in Vps18 KO (59%) com-
pared with WT (17%) cells, whereas the percentage in the TGN was
strikingly decreased (39% in WT to 18% in Vps18 KO, Figure 5E).
Double labeling of CI-MPR with SNX1 showed that in HeLa WT cells
58% of the SNX1 endosomal tubules were also positive for CI-MPR
(Figure 5A and C, arrowheads, F). In HOPS KO cells, SNX1-positive
vesicles and tubules were still readily found near endosomes/HOPS
bodies (Figure 5D; Supplementary Figure S6, blue pseudocolor),
but the percentage that also contained CI-MPR had decreased to
38% (Figure 5D and F; Supplementary Figure S6).
The ndings in Figures 4 and 5 and Supplementary Figure S6
altogether demonstrated that CI-MPR was depleted from the TGN
and redistributed to endosomes upon HOPS KO. We still found
SNX1-positive recycling tubules on endosomes in HOPS KO cells,
but CI-MPR failed to efciently enter these. Impairment of retro-
grade transport to the TGN explained the relative shift in CI-MPR
localization from TGN toward endosomes.
HOPS bodies are not acidified
What could prevent the CI-MPR from entering SNX1 recycling tu-
bules? Retrograde transport of CI-MPR presumably depends on li-
gand dissociation, which allows CI-MPR to engage in further rounds
of hydrolase sorting at the TGN (Brown etal., 1984; Brown etal.,
1986; Braulke and Bonifacino, 2009). Dissociation between acid
hydrolases and CI-MPR is induced by acidication of maturing
endosomes (Brown et al., 1986; Olson et al., 2020). To test if the
impairment of CI-MPR recycling could be caused by an acidication
defect of HOPS bodies, we treated HeLa WT and Vps18 KO cells
for 3 h with Chloroquine or Balomycin A1. Chloroquine is a weak
base that accumulates and neutralizes acidied compartments
(Homewood etal., 1972), while Balomycin A1 blocks the V-ATPase,
preventing endo-lysosomal acidication (Bowman et al., 1988;
Yoshimori et al., 1991). In HeLa WT cells, both drugs caused a
statistically signicant redistribution of CI-MPR to EEA1-positive en-
dosomes (Figure 6A-B), which is in agreement with previous studies
(Brown etal., 1984; Brown etal., 1986; van Weert etal., 1995). In
contrast, in Vps18 KO cells we found no additional effect of the
drugs on CI-MPR redistribution. These data implied that impaired
retrograde recycling of CI-MPR in HOPS KO cells could be due to a
lack of acidication of HOPS bodies.
To assess the pH of HOPS bodies, we performed live-cell imaging
of cells incubated for 3 h with Dextran-uorescein. Fluorescein uo-
rescence is pH-sensitive under excitation with 480 nm but not 440 nm
light, making it a robust ratiometric pH sensor (Canton and Grinstein,
2015). To address HOPS bodies specically, and not all endocytic
compartments, we imaged HeLa WT, Vps18, and Vps39 KO cells
stably expressing EEA1-mCherry. We rst calibrated our uores-
cence readout by imaging pH-clamped HeLa WT cells (see Materials
and Methods; Supplementary Figure S7) and then imaged the EEA1-
mCherry−expressing lines in normal culture medium. We found that
the ratio of uorescein 440/480 intensity in the EEA1-positive com-
partments corresponded to an average pH of 5.7 in WT cells and 5.9
in both Vps18 and Vps39 KO cells (Figure 6C and D). This demon-
strated that the HOPS bodies, though they acquire late endosomal
characteristics, undergo only mild acidication typical for early stages
of the endo-lysosomal system (Huotari and Helenius, 2011).
Overall, these data indicated that a key aspect of late endosomal
maturation – that is, acidication – is impaired in HOPS bodies.
Because a lack of acidication prevents CI-MPR–ligand dissociation,
the near neutral pH in HOPS bodies is a likely explanation for the
observed accumulation of CI-MPR.
HOPS bodies have a mixed early and late morphology and
receive endocytic cargo
EM is the method of choice to link molecular composition to cellular
ultrastructure and has been exceptionally insightful in characteriza-
tion of the endo-lysosomal system (Klumperman and Raposo, 2014).
The immuno-EM of CI-MPR and SNX1 (Figure 5; Supplementary
Figure S6) already revealed endo-lysosomal organelles of peculiar
morphology, presumably representing the HOPS bodies. Unfortu-
nately, our marker to label HOPS bodies by IF, EEA1, labels poorly
in immuno-EM (van der Beek etal., 2022), hampering correlation
between our IF and EM data. To overcome this, we used our re-
cently developed on-section CLEM method to localize proteins in
EM based on their IF staining (van der Beek etal., 2022).
HeLa WT and Vps18 KO cells were incubated with the endocytic
tracer bovine serum albumin conjugated to 5-nm gold particles
(BSA5) for 3 h, xed with 4% formaldehyde, and prepared for cryo-
sectioning. Ultrathin cryosections were uorescently labeled and
scanned by uorescence microscopy for EEA1-positive compart-
ments. Then the same sections were transferred to EM and corre-
lated to the IF signal. In EM, distinct types of endo-lysosomal com-
partments were dened by their morphology (see Materials and
Methods for detailed denitions). Consistent with our previous nd-
ings (van der Beek etal., 2022), in HeLa WT cells EEA1 was present
on early endosomes (electron lucent vacuoles partially coated with
clathrin and displaying associated tubules) (Figure 7A), as well as
late endosomes (rounded to oval shaped organelles with ≥6 ILVs)
(Figure 7A). Lysosomes (small-sized compartments with amorphous
electron-dense content and sometimes “onion-like” concentric
membranes) generally lacked EEA1. In HOPS KO cells, EEA1 also
localized to “classical” early and late endosomes, but additionally
associated with numerous enlarged compartments of complex ul-
trastructure, the HOPS bodies (Figure 7B and D−F; Supplementary
Figure S8, A−E). Quantitation of the relative distribution of EEA1 in
HeLa WT versus Vps18 KO cells showed a signicant redistribution
of EEA1 label to the HOPS bodies (Figure 7C), in line with the IF
data (Figure 2). The EEA1-positive HOPS bodies were identical to
the compartments in which we found accumulation of CI-MPR
(Figure 5; Supplementary Figure S6)
EM analysis of the HOPS bodies disclosed them as large vacu-
oles that regularly displayed clathrin coats at the cytosolic face of
their limiting membrane and associated tubules that extended from
the vacuolar part. The clathrin coat is indicative for the presence of
ESCRT and ongoing ILV formation, whereas the tubules mediate
recycling, which are both features of early endosomes (Raiborg
etal., 2002; Klumperman and Raposo, 2014). In addition, the lumen
of the HOPS bodies exhibited numerous ILVs, membrane whorls,
and electron-dense material, which are characteristic of late endo-
somes-lysosomes. Furthermore, HOPS bodies regularly contained
endocytosed BSA5 and amorphous, granular material indicative of
lipid accumulation. This is consistent with previous ndings from
(Anderson etal., 2022), who found an endo-lysosomal accumulation
of cholesterol upon KO of Vps41 and Vps39.
We next xed HeLa WT and various HOPS KO cells by high-
pressure freezing (HPF) for resin EM. HPF xation is less appropriate
for immuno-EM, but the prime approach for purely ultrastructural
studies. Moreover, the high contrast and large size of resin EM sec-
tions makes them optimal for systematic morphological analyses.
After HPF xation, HOPS bodies were readily recognized as enlarged
vacuoles containing a mix of membranes, amorphous content and
ILVs and displaying clathrin coats and associated tubules (Figure
8D−G). Strikingly, in low magnication overviews we found in all
HOPS KO cells an accumulation of HOPS bodies as well as an overall
10 | J. van der Beek et al. Molecular Biology of the Cell
FIGURE 5: CI-MPR does not enter SNX1-positive tubules in HOPS KO cells. Immuno-EM of SNX1 (15 nm gold) and
CI-MPR (10 nm gold) in HeLa WT (A, C) and Vps18 KO (B, D) cells. In (A) and (D), endocytic tracer BSA5 was added to
cells 3 h before fixation. SNX1-positive tubules are highlighted in blue. In WT cells SNX1-positive tubules contain
CI-MPR (arrows). In Vps18 KO cells CI-MPR accumulates in the lumen of the HOPS bodies (B, asterisks), despite the
presence of associated SNX1-positive tubules (D, highlighted blue). See Supplementary Figure S6 for additional pictures
and other HOPS KO cells. (E) Quantification of CI-MPR immunogold labeling. CI-MPR is depleted from TGN in Vps18
KO cells. Within endosomes, CI-MPR is enriched in both the limiting membrane and lumen. n > 300 gold particles per
condition. (F) Quantification of the percentage of SNX1-positive tubules also positive for CI-MPR labeling, n = 58 and
105 SNX1-positive tubules for WT and Vps18 KO, respectively. *, HOPS body; G, Golgi; M, mitochondrion; LE, late
endosome; Ly, lysosome. Scale bars 200 nm.
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 11
FIGURE 6: Elevated pH inhibits retrograde CI-MPR transport from HOPS bodies. (A) IF of CI-MPR and EEA1 in HeLa WT
and Vps18 KO cells, untreated or treated with Bafilomycin A1 (Baf) or Chloroquine (CQ) for 3 h. (B) Quantification of the
percentage of thresholded EEA1 signal overlapping with CI-MPR signal. n ≥ 24 for each condition. In Baf- or CQ-treated
HeLa WT cells CI-MPR disperses to EEA1-positive compartments to the same level as in nontreated Vps18 KO cells. In
Vps18 KO cells treatment has no additional effect. (C) Live-cell fluorescence imaging of WT, Vps18 and Vps39 KO cells
expressing EEA1-mCherry, incubated for 3 h with Dextran-fluorescein. The ratio of 440/480 excitation of fluorescein
serves as a pH sensor. This ratio is translated to actual pH using calibration samples described in Supplementary Figure
S7. Several examples of individual pH measurements on compartments are shown. (D) Quantification of the 440/480
ratios found in EEA1 compartments of HeLa WT, Vps18, and Vps39 KO cells. n = 324, 310, and 68 EEA1 compartments
from ≥ 10 cells from WT, Vps18, and Vps39 KO, respectively. Changes between conditions are significant at p < 0.01 (**)
or p < 0.001 (***) or not significant (n.s.), by Kruskal−Wallis multiple comparison test. Dots in dot plots represent
individual cells (B) or single EEA1 compartments (D). Scale bars 20 µm (overviews), 5 µm (insets).
12 | J. van der Beek et al. Molecular Biology of the Cell
3
1
2
1
3
2
HeLa WT
EEA1
Vps18KO
EEA1
N
N
2
1
2
13
3
EE
M
M
M
EE
*
*
*
EE
LE
Morphological classication of
EEA1-positive organelles
WT Vps18KO
0%
20%
40%
60%
80%
100%
Early endosomes Late endosomes
Lysosomes HOPS bodies
HeLa Vps11KO
EEA1
HeLa Vps39KO
EEA1
HeLa Vps41KO
EEA1
N
N
*
***
B
CDEF
A
L
E
EE
BSA5 3h
BSA5 3h
BSA5 3hBSA5 3hBSA5 3h
FIGURE 7: HOPS bodies display early and late ultrastructural characteristics. (A) CLEM of EEA1 in HeLa WT cells. EEA1
localizes to early (A1, A3) and late (A2) endosomes. Early endosomes were defined by their electron-lucent lumen,
sorting tubules (closed arrows) and clathrin coats (open arrows). Late endosomes by numerous ILVs and dense content.
(B) CLEM of EEA1 in Vps18 KO cells. EEA1 localizes to early endosomes (B1) and large, hybrid early-late endosomal
compartments with heterogeneous content and clathrin coats (open arrow, B2, B3), that is, HOPS bodies (asterisks).
(C) Classification of EEA1-positive organelles based on ultrastructure. In Vps18 KO cells, EEA1 label is highly enriched
over HOPS bodies with hybrid early-late ultrastructure. n = 25 and 48 organelles for WT and Vps18 KO, respectively.
(D−F) CLEM of EEA1 in HeLa Vps11 (D), Vps39 (E), and Vps41 (F) KO cells invariantly localizes EEA1 to HOPS bodies.
*, HOPS bodies; EE, early endosome; M, mitochondrion; N, nucleus; LE, late endosome. 1 µm scale bars in CLEM
overview, 200 nm in EM insets.
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 13
increase in endo-lysosomal organelles (Figure 8;Supplementary
Figure S9). This prompted us to analyze the relative composition of
the endo-lysosomal system for each condition. We randomly
screened for ∼200 endosomal compartments and classied these by
morphology (Figure 8H). This revealed a minor relative decrease of
early endosomes (39% in WT, 12−28% in HOPS KO cells) and lyso-
somes (49% in WT, 33−47% in HOPS KO cells) and a relative in-
crease in late endosomes (4% in WT, 10−12% in HOPS KO cells). The
fraction of HOPS bodies drastically increased in all HOPS KO cells
(8% in WT, 29−41% in HOPS KO cells), conrming that accumulation
of these organelles is specic for HOPS dysfunction (Figure 8H).
We also analyzed the distribution of endocytosed BSA5. In WT
cells, endocytosed BSA5 was found in early and late endosomes as
well as lysosomes. In HOPS KO cells, early and late endosomes and
HOPS bodies were positive for BSA5, but lysosomes were consis-
tently negative (Figure 8I). The presence of endocytosed BSA5 in
HOPS bodies was consistent with the uorescent data on internal-
ized Dextran-AF488 (Figure 1C and D). The absence of BSA5 from
“classical” lysosomes conrmed previous EM studies in chemically
xed Vps41-silenced or -KO cells (Pols etal., 2013a; van der Welle
etal., 2021).
Together, these EM data showed that all HOPS KO cells showed
a striking increase in HOPS bodies, indicating that these organelles
specically accumulate upon HOPS dysfunction. HOPS bodies dis-
played a typical hybrid early−late endosomal morphology, which is
in agreement with their hybrid early−late molecular composition
(Figures 1 and 2). Interestingly, the BSA5 uptake studies positioned
early endosomes, late endosomes and HOPS bodies upstream of
the HOPS dependent fusion step and lysosomes downstream.
These data highlighted the role of HOPS as a determining factor for
the transfer of endocytosed cargo from endosomes to lysosomes,
while upstream of this transport step HOPS KO causes the accumu-
lation of endosomes of hybrid early−late morphology.
HOPS bodies contain p62/LC3-positive autophagic content
The current model of macroautophagy is that autophagosomes
fuse directly with lysosomes to form autolysosomes, or with endo-
somes resulting in amphisomes that subsequently fuse with lyso-
somes (Yu etal., 2018; Hu and Reggiori, 2022). These fusions have
been reported to depend on the HOPS complex (Jiang etal., 2014;
Takáts etal., 2014). Consistent with this, previous studies in HOPS
depleted cells (van der Welle et al., 2021; Terawaki et al., 2023)
showed increased levels of lipidated LC3 (LC3-II), the form of LC3
that is conjugated onto the autophagosomal membrane during
macroautophagy. Higher levels of LC3-II are often interpreted as an
increase in early autophagic compartments that are not (yet) fused
with lysosomes. By Western blot, we conrmed the accumulation
of LC3-II in all our HOPS KO cells, including those in A549 and
HT1080 lines (Supplementary Fig. S10A). However, EM of HOPS
KO cells did not reveal a marked increase in typical autophago-
somes (Figure 8). Instead, we observed granules inside the HOPS
bodies that were morphologically reminiscent to autophagic con-
tent accumulating in endo-lysosomes when cells are starved in the
presence of the V-ATPase inhibitor Balomycin A1 (De Mazière
etal., 2022). These 100−250 nm sized, amorphous dense granules
were frequently membrane-bound (Figures 7 and 8) and observed
in all HOPS KO cells, both after HPF and chemical xation. To es-
tablish or disprove the presence of autophagic content in HOPS
bodies, we applied our recently optimized labeling protocol for
LC3 and p62 immuno-EM (De Mazière etal., 2022). This unequivo-
cally localized p62 and LC3 in the lumen of HOPS bodies, in which
they associated with the typical dense granules (Figure 9A and B,
arrows). Of note, p62 and LC3 labeling was consistently absent
from lysosomes, which – as shown above –also lacked internalized
BSA5 (Figure 8I).
We then performed immuno-EM of p62 combined with BSA5
uptake (3 h), screened randomly for BSA5-positive endosomes and
established the percentage also labeled for p62. In WT control
cells p62 is rapidly degraded in endo-lysosomal compartments
and indeed <5% of BSA5-positive endo-lysosomes contained p62
label. In contrast, in Vps41 KO cells most (60%, Figure 9D) BSA5-
positive compartments contained p62 and virtually all of these
double-positive compartments were HOPS bodies. These data
indicated that incorporation of autophagic cargo into HOPS bod-
ies proceeded in the absence of HOPS. To investigate if this was
dependent on macroautophagy we induced silencing of ATG7, a
key protein for early stage autophagosome formation (Mizushima,
2020). By siRNA, we reached a depletion of ≥80% as measured by
QPCR. This caused a striking increase in cytoplasmic patches of
p62 in both HeLa WT and Vps41 KO cells (Figure 9C). Interest-
ingly, despite the increase pf cytosolic p62, incorporation into
HOPS bodies of Vps41 KO cells was strikingly reduced by deple-
tion of ATG7 (from 60% in control siRNA to 41% in ATG7 siRNA,
Figure 9C and D). These data indicated that the autophagic mate-
rial in Vps41 KO endosomes at least in part derived from macroau-
tophagy and implied that autophagosome-endosome fusion, that
is, formation of amphisomes, proceeded in the absence of HOPS.
To further investigate this, we depleted the autophagosomal
SNAREs Syntaxin 17 or Ykt6, which are part of the same fusion
machinery as the HOPS complex (Jiang etal., 2014; Takáts etal.,
2014; ; Bas et al., 2018; Takáts et al., 2018). Depletion of these
SNAREs in Vps41 KO cells did not decrease incorporation of p62
in BSA5-positive compartments (Figure 9D; Supplementary
Figure S10B). This further supported that macroautophagy of p62
into HOPS bodies could occur independent of the HOPS-Syntaxin
17-Ykt6 fusion complex.
Invagination of the endosomal membrane relies on the ESCRT
complex (Vietri etal., 2020). ESCRT-III has a major role in conven-
tional ILV formation, but can also internalize cytosolic content marked
by p62 for degradation in endo-lysosomes (Mejlvang etal., 2018).
This process is called microautophagy and relies on ESCRT compo-
nents CHMP4B, Vps4A and Vps4B. Because HOPS bodies displayed
typical bi-layered clathrin coats known to contain ESCRT compo-
nents, we asked if microautophagy could contribute to the HOPS-
independent uptake of p62 in HOPS bodies. In Vps41 KO cells de-
pleted for CHMP4B or Vps4A/B; however, the HOPS bodies
contained similar levels of p62 as in nondepleted cells (63% and 66%
of BSA5-positive organelles, Figure 9C and D). These data indicated
that p62/autophagic content did not enter HOPS bodies through
microautophagy (Figure 9C and D; Supplementary Figure S10B).
Strikingly, Vps4A/B depletion led to an increase of p62 at the limiting
membrane of HOPS bodies, indicative of lysophagy. Vps4A/B deple-
tion also led to a dramatic cell death in Vps41 KO but not WT cells.
These data identied HOPS bodies as amphisomes. Incorpora-
tion of p62 and LC3 in HOPS bodies required ATG7-dependent
autophagosome formation, but was independent of the HOPS-Syn-
taxin 17-Ykt6 complex for subsequent autophagosome−endosome
fusion. In short, our results implied that amphisome formation, but
not autolysosome formation, is independent of HOPS.
DISCUSSION
In this paper, we addressed the role of the HOPS complex in the
functional organization of the endo-lysosomal system. By generat-
ing HeLa KO cell lines for 4 different HOPS subunits, the class C core
14 | J. van der Beek et al. Molecular Biology of the Cell
Relative counts of endo-lysosomal organelles Percentage of organelles reached by BSA5
WT 11KO 18KO 39KO 41KO
0%
20%
40%
60%
80%
100%
Early endosomes Late endosomes
Lysosomes HOPS bodies
Early
endosomes
Late
endosomes
HOPS
bodiesLysosomes
0%
20%
40%
60%
80%
100%
WT 11KO 18KO 39KO 41KO
A
HeLa WT
C
B
D
E
HI
FG
HeLa Vps18KO
HeLa WT BSA5 3h
BSA5 3h
BSA5 3hBSA5 3hBSA5 3h
HeLa Vps18KO
HeLa Vps11KO HeLa Vps39KO HeLa Vps41KO
N
N
N
EE
G
G
Ly
Ly
Ly
LE
Ly
Ly
EE
*
*
*
*
**
*
*
*
*
**
*
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 15
Vps11 and Vps18 and the HOPS-specic Vps39 and Vps41, we
monitored the general defects of HOPS depletion. In all KO cell
lines, we found a profound effect on prelysosomal compartments,
as illustrated by increased colocalization of early (Rab5, PtdIns3P,
EEA1) and late (Rab7, CD63, LAMP1, cathepsin D) endosomal mark-
ers (Figures 1 and 2; Supplementary Figures S1 and S2). By EM,
HOPS KO cells showed an accumulation of morphologically hybrid
compartments with ultrastructural characteristics of early (clathrin
coats, recycling tubules) and late (ILVs, heterogenous content) en-
dosomes (Figures 7 and 8; Supplementary Figures S8 and S9), to
which we referred to as HOPS bodies. HOPS bodies were only
mildly acidic (Figure 6C and D), were reached by endocytosed cargo
(Figure 8) and unexpectedly also by autophagic proteins (Figure 9),
which dened them as amphisomes. HOPS bodies also accumu-
lated CI-MPR, the receptor for lysosomal enzymes that normally cy-
cles between TGN and endosomes. CI-MPR failed to enter SNX1-
positive retrograde recycling tubules originating from HOPS bodies,
leading to receptor depletion from the TGN. Concomitantly, the
levels of inactive pro-cathepsin D, a lysosomal hydrolase and sub-
strate for CI-MPR, increased in the Golgi/TGN and in the medium
(Figure 3; Supplementary Figures S3 and S5C). Cathepsin D that did
reach HOPS bodies was not processed to its mature form and re-
mained inactive. Together, these data show that loss of HOPS com-
plex blocks or delays the transition of early to late endosomes, af-
fects endosomal recycling properties and alters lysosomal enzyme
trafcking and activation. Further, they suggest the existence of a
HOPS-independent pathway for endosome-autophagosome fu-
sion. Our ndings are summarized in a model (Figure 10).
A striking phenotype of all HOPS KO cells was the accumulation
of large, Rab5-Rab7−positive hybrid endosomes, the HOPS bodies
(Figure 1, and E and F). The hybrid molecular make-up, increase
in size, perinuclear localization, as well as the overall ultrastructure
of these HOPS bodies resembled the endo-lysosomal phenotype
of cells overexpressing constitutively active Rab5 (Rab5Q79L)
(Wegener etal., 2010), suggesting that lack of HOPS induced a de-
fect in Rab5 to Rab7 conversion. Likewise, early studies in live cells
have shown that depletion of the HOPS complex delays Rab5-to-
Rab7 conversion (Rink etal., 2005) and more recently others found
that HOPS interacts with Rab5, Rab7, several of their effectors
(Gillingham etal., 2014; Van Der Kant etal., 2015; Jongsma etal.,
2020) and the Mon1−Ccz1 complex, the Rab7 GEF required for
Rab5 to Rab7 transition (Wang etal., 2003; Poteryaev etal., 2010).
Based on these ndings, it was suggested that subunits of the HOPS
complex could act as a molecular platform for the Rab5-Rab7 con-
version process. However, contradicting in vitro data showed that
the HOPS complex is dispensable for Rab5 to Rab7 conversion
(Langemeyer etal., 2020). In our study, knock-out of any single sub-
unit resulted in increased Rab5-Rab7 colocalization. This indirect
readout for Rab5 to Rab7 conversion did not reveal the precise role
of HOPS in this process. Nonetheless, it indicated that all subunits
are required for normal Rab5 to Rab7 transition. If HOPS would act
as a molecular platform for this conversion, we would have expected
to see differences between the loss of specic subunits, with stron-
ger effects for depletion of those subunits that facilitate key interac-
tions. Based on our data, we consider it more probable that the role
of HOPS in Rab5 to Rab7 conversion depends on membrane fusion
activity of the full complex.
A surprising nding was the presence of p62 and LC3 in HOPS
bodies (Figure 9), which dened them as amphisomes. Of note, ex-
pression of Orf3A, a protein encoded by SARS-CoV-2 that binds
Vps39 and inactivates HOPS, also causes the accumulation of
amphisomes (Miao etal., 2021). Furthermore, accumulation of p62
in cells was noted after downregulation of Vps41 by exposure to
Cadmium (Wang et al., 2023). The accumulation of p62 in HOPS
bodies is potentially relevant with respect to HOPS disease develop-
ment (van der Beek etal., 2019; Monfrini etal., 2021b), since defects
in p62-mediated proteostasis are linked to several types of neurode-
generative diseases (Kumar etal., 2022). Both p62 and LC3 were, by
immuno-EM, associated with typical dense granules indicative for
the presence of autophagic cargo, as previously observed in cells
treated with Balomycin A1 (De Mazière etal., 2022). Because HOPS
bodies were not or mildly acidic and only contained inactive pro-ca-
thepsin D, the accumulation of p62- and LC3-marked autophagic
cargo in their lumens is explained by this lack of degradative capac-
ity. p62 and endocytosed BSA5 were both found in HOPS bodies,
but absent from lysosomes. Hence, in the absence of HOPS both
endocytic and autophagic cargo reached endosomal compartments,
but transfer to lysosomes was inhibited. Previously, using the same
denitions for endosomes and lysosomes as here, we found that ly-
sosomes are the major sites for hydrolase activity (Liv et al., 2023).
Altogether this suggests that HOPS is required for the transfer of
endocytic and autophagic cargo from predominantly non-active en-
dosomes to actively degrading lysosomes, signifying HOPS as an
important gate keeper for lysosomal entry and cargo degradation.
The formation of amphisomes in HOPS KO cells implied that
endosomal delivery of autophagic cargo proceeded in the absence
of HOPS. Likewise, depletion of the SNAREs Syntaxin 17 or Ykt6,
both implicated in HOPS-dependent autophagosome – endo-lyso-
some fusion (Lürick etal., 2015; Matsui etal., 2018; Takáts etal.,
2018), did not block entry of p62 in HOPS bodies (Figure 9C-D). This
was unexpected since it has been reported that autophagic ux de-
pends on the interaction between these autophagosomal SNAREs
and the HOPS complex (Jiang et al., 2014; Takáts et al., 2014).
Knockdowns of Vps4A/B or CHMP4B (Mejlvang et al., 2018) in
FIGURE 8: HOPS KO induces accumulation of hybrid early – late endosomes, that is, HOPS bodies. HeLa WT and KO
cells incubated with BSA5 for 3 h were HPF fixed and embedded in EPON for EM imaging. (A, B) Overview of endocytic
organelles in HeLa WT cells. Early endosomes (EE) show characteristic associated tubules and clathrin coats
(arrowheads). Lysosomes (Ly) are small and dense and regularly positive for endocytosed BSA5. (C, D) Vps18 KO cells.
(C) Note the high abundance of endocytic compartments. (D) HOPS bodies (asterisks) appear as enlarged, BSA5-positive
endosomal organelles with hybrid early and late endo-lysosomal features: associated vesicles and tubules, clathrin coats
(arrowheads), membrane whorls, dense content, numerous ILVs. (E−G) Examples of HOPS bodies (asterisks) in Vps11,
Vps39 and Vps41 KO cells. Lysosomes are mostly negative for BSA5. For more examples see Supplementary Figure S9.
(H) Classification of endosomal organelles in HeLa WT and KO cells. HOPS bodies are strongly enriched in HOPS KO
cells. n ≥ 200 organelles per condition. (I) Percentage of organelles containing BSA5. BSA5 reaches 67% of lysosomes in
WT cells and only ∼10% in HOPS KO cells. n ≥ 200 organelles per condition. *, Hybrid organelle; EE, early endosome; G,
Golgi; N, nucleus; Ly, lysosome. Scale bars 2 µm (A, C), 200 nm (B, D−G).
16 | J. van der Beek et al. Molecular Biology of the Cell
*
*
*
*
*
*
*
**
*
A
B
HeLa WT
LAMP110
LC315
HeLa Vps11KO HeLa Vps18KO HeLa Vps39KO HeLa Vps41KO
HeLa WT
LAMP115
p6210
BSA5
C
D
HeLa Vps11KO HeLa Vps18KO HeLa Vps39KO HeLa Vps41KO
N
Ly Ly
LE Ly
Ly
Ly
Ly
Ly
M
siVps4A/BsiATG7
siVps4A/BsiATG7
HeLa WT
p6210
BSA5
HeLa Vps41KO
p6210
BSA5
WT
no p62
luminal p62
CHMP4B
Ykt6
Vps41KO
Percentage of BSA-positive organelles with internal p62
0%
20%
40%
60%
80%
100%
Scr
Vps4A/B
Stx17
ATG7
Scr
Vps4A/B
CHMP4B
Ykt6
Stx17
ATG7
*
*
Ly
siStx17
siStx17
*
EE EE
LE
FIGURE 9: HOPS bodies contain autophagic materials. Cells were prepared for immuno-EM as in Figure 5. (A) Double
immunogold labeling of LC3 (10 nm) and LAMP1 (15 nm). LC3 labels the typical dense structures (arrowheads) within
LAMP1-positive HOPS bodies (asterisks). In WT cells, LC3 is sometimes seen on cytoplasmic granules (arrow), but not in
endosomes. (B) Double labeling of p62 (10 nm) and LAMP1 (15 nm) in WT and HOPS KO cells. In WT cells, p62 label is
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 17
Vps41 KO cells excluded that uptake of p62 in HOPS bodies de-
pended on microautophagy (Figure 9C and D). In contrast, deple-
tion of ATG7–required for autophagosome formation–reduced the
number of p62-positive endosomes from 60% to 41%. Together
these data inferred that at least some of the autophagic cargo pres-
ent in HOPS bodies derived from macroautophagy (Figure 9C and
D), implying the existence of an alternative, HOPS-Stx17-Ykt6 inde-
pendent mechanism of autophagosome –endosome fusion. The ac-
cumulation of HOPS bodies in HOPS KO cells is in line with the
scenario that subsequent amphisome–lysosome fusion is HOPS de-
pendent. Interestingly, the high numbers of HOPS bodies raise the
question if autophagosome–endosome fusion normally prevails
over autophagosome–lysosome fusion. Formation of amphisomes
in control conditions may be obscured by rapid turnover of the am-
phisomes. The current literature on amphisome and autolysosome
formation is rather ambiguous and clearly more work is required to
address these questions (Reggiori and Ungermann, 2017; Ganesan
and Cai, 2021; Hu and Reggiori, 2022).
Another unexpected nding was that depletion of the HOPS
complex caused a redistribution of CI-MPR from the TGN to HOPS
bodies (Figure 4). Depletion of CI-MPR from the TGN is known to
increase secretion of lysosomal enzyme precursors (Meel and
Klumperman, 2014), which was indeed the case for HOPS KO cells
(Supplementary Figure S5C). Likewise, Anderson et al. recently re-
ported a decrease in cellular CI-MPR levels in Vps39 or Vps41 KO
cells and an increased secretion of CI-MPR ligand NPC2 (Anderson
etal., 2022). Lack of NPC2 causes an accumulation of endo-lyso-
somal cholesterol (Anderson etal., 2022), this was supported by the
lipid buildup we noticed in our EM studies (Figure 6) and was
reported earlier (Swetha etal., 2011). These and our present studies
all indicate a trafcking defect for CI-MPR in HOPS KO cells.
Since we observed that CI-MPR accumulated in HOPS bodies, we
investigated if the SNX1-positive tubules of the retrograde ESCPE
pathway were still formed. All our HOPS KO cell lines readily dis-
played SNX1-positive tubules by both IF and by immuno-EM
(Figures 4A and B, 5, A−D; Supplementary Figure S6). In HOPS KO
only found in the cytoplasm (arrowhead). In HOPS KO cells, p62 is found in the HOPS bodies (asterisks) on similar
structures as LC3 (arrowheads). (C) HeLa WT and Vps41 KO cells depleted for ATG7 (macroautophagy), Vps4A/B
(microautophagy) or Stx17 (SNARE autophagosome−lysosome fusion) and labeled for p62 (10 n). Note accumulation of
p62 on limiting membrane of HOPS bodies in Vps4A/B-depleted Vps41 KO cells, indicative of lysophagy. For more
representative images and depletion conditions see Supplementary Figure S10B. (D) Quantification of the % BSA5-
positive organelles that contain p62 label. Only depletion of ATG7 reduces the amount of p62-positive HOPS bodies in
Vps41 KO cells. >50 BSA5-positive organelles were scored in each condition. EE, early endosome; N, nucleus; LE, late
endosome; Ly, lysosome. Scale bars, 200 nm.
FIGURE 10: Summarizing model on the effects of HOPS depletion on the functional organization of the endo-lysosomal
system. Loss of the HOPS complex causes accumulation of HOPS bodies, organelles of hybrid molecular and
morphological composition. HOPS bodies are reached by endocytic and autophagic cargoes but fail to fuse with
lysosomes. They have low enzymatic activity and are of neutral pH, which prevents degradation of endocytic and
autophagic content in HOPS bodies, as well as the retrograde transport of CI-MPR to the Trans-Golgi network.
Depletion of CI-MPR from TGN leads to local accumulation of pro-cathepsin D and a general increase in secretion of
lysosomal hydrolases and autophagic content from HOPS bodies.
18 | J. van der Beek et al. Molecular Biology of the Cell
cells, however, these tubules were generally depleted from CI-MPR
(Figure 5D), suggesting a block in the entry of CI-MPR into ESCPE
tubules rather than a general block in recycling (Evans etal., 2020;
Simonetti etal., 2023). In early studies it was shown that CI-MPR-li-
gand dissociation in acidifying endosomes is both necessary and
sufcient to trigger recycling of CI-MPRs to the TGN (Brown etal.,
1986). Concomitantly, we found that inhibition of endosomal acidi-
cation by Chloroquine or Balomycin A1 relocalized CI-MPR from
TGN to endosomes in HeLa WT cells. Interestingly, the drugs had no
additional effect on CI-MPR redistribution in HOPS KO cells (Figure
6A and B). Moreover, by uorescein pH measurements we found
that HOPS bodies were not or only mildly acidied (Figure 6C and
D). This lack of acidication is a plausible explanation for CI-MPR
retention in the HOPS bodies and also explains why lysosomal en-
zymes in HOPS bodies are not processed to their active form
(Figure 3). Of note, previous studies from our lab and others failed to
nd defects in CI-MPR and lysosomal enzyme transport after knock-
down of HOPS subunits (Swetha et al., 2011; Pols et al., 2013a).
Also, the “HOPS bodies” were not reported before, although we
previously characterized Vps39 or Vps41-depleted cells by EM (Pols
etal., 2013a). The main difference between these and our present
study is the use of genetic editing instead of siRNA-based silencing.
From this we conclude that the accumulation of HOPS bodies and
the endosomal trafcking defects occurs only after complete and
long-term removal of HOPS complex activity. Altogether, we here
showed that HOPS KO severely affected CI-MPR transport and
thereby the trafcking and activation of lysosomal enzymes.
Interestingly, we did not nd any obvious differences between
the four different KO cell lines, even though Vps11 and Vps18 are
also part of sister complex CORVET. This consistency in phenotypes
was reported before (Wartosch et al., 2015). Similarly, in a recent
overview of CORVET and HOPS related diseases, we noticed that
most pathological lesions caused by mutations in core components
were related to the later stages of the endo-lysosomal system (van
der Beek etal., 2019). This makes it plausible that the phenotypes
described in this study depend on assembly and activity of the full
HOPS complex. We can, however, not rule out that depletion of in-
dividual subunits additionally affects specic subsets of endosomes
or trafcking pathways, such as APPL1 endosomes or specic recy-
cling pathways (Perini etal., 2014; Jonker etal., 2018), that were not
investigated in this study.
The loss of endosome – lysosome fusion induced by HOPS KO
cells does not easily explain the maturation, recycling and acidica-
tion defects in HOPS bodies, which occur upstream of lysosomes. A
possible explanation is that the HOPS complex is also required for
fusion of TGN-derived vesicles carrying lysosomal membrane pro-
teins to late endosomes (Pols et al., 2013b; Davis et al., 2021). In
yeast this pathway is known as the ALP pathway, which delivers lyso-
somal membrane proteins directly to the vacuole (Cowles et al.,
1997; Anand etal., 2009; Llinares etal., 2015). In Drosophila, a similar
Vps41/Light−dependent TGN-derived pathway is important for lyso-
somal delivery of the membrane proteins LAMP1, NPC1, and the
V-ATPase (Swetha et al., 2011). Indeed, we found small vesicles ac-
cumulating around the HOPS bodies in Vps41 KO cells (Figure 8G;
Supplementary Figure S9E), which were morphologically reminiscent
of the TGN-derived LAMP carriers previously identied in Vps41 and
VAMP7 knockdown cells (Pols etal., 2013b; Davis etal., 2021), and
positive for LAMP1 by immuno-EM (Pols etal., 2013b). A lack in the
delivery of lysosomal membrane proteins could directly and indi-
rectly affect endosomal maturation, for example by altering recruit-
ment of phosphoinositides or effector proteins, by altering choles-
terol processing and egress from endosomes, changing pH or ionic
balance, and by activation of lysosomal hydrolases (Huotari and
Helenius, 2011). Further experimentation is required to investigate
and proof this hypothesis. Interestingly, HOPS KO cells still contained
active, acidied lysosomes (Wartosch et al., 2015; van der Welle
etal., 2021), albeit that these were not reached by endocytosed and
autophagic cargo in our experimental conditions. In fact, HOPS KO
cells showed a general increase in endo-lysosomal compartments
(Figure 8H), which is likely explained by nuclear translocation of
TFE3/TFEB and consequent activation of the CLEAR network (van
der Welle et al., 2021). These observations might also explain why
some studies report an effect on hydrolase activity after HOPS deple-
tion, for example in D. melanogaster car/Vps33A or dor/Vps18 mu-
tants and Vps18 conditional KO mice (Sriram etal., 2003; Peng etal.,
2012), while others do not (Swetha etal., 2011; Graham etal., 2013;
Pols etal., 2013b; van der Welle etal., 2021;). How and by which ki-
netics lysosomes in HOPS KO cells are formed and how they might
differ from lysosomes in WT cells remains a topic for future studies.
Summarizing, our data show that HOPS complex is required for
endosomal maturation prior to the stage of endo-lysosomal fusion
and reveal a central role of the HOPS tethering complex in orches-
trating the endo-lysosomal system, including endosomal recycling
pathways and autophagic clearance. Moreover, we provide evi-
dence for a HOPS-independent pathway for incorporation of au-
tophagic material into endosomes, which evokes further investiga-
tion into the role and mechanism of amphisome formation.
MATERIALS AND METHODS
Request a protocol throughBio-protocol.
Antibodies and reagents
For antibodies used in this study, see (Table 1), effectene transfection
reagent (301425, QIAGEN) and HiPerFect transfection reagents
(301704, QIAGEN) were used. SiRlysosome (SC012, Spirochrome),
Dextran-AF488 (D22910, Thermo Fisher Scientic) and Dextran-
uorescein (D1820, Thermo Fisher Scientic) were used in uores-
cence microscopy. The following siRNAs were used: All stars nega-
tive control siRNA (1027281, QIAGEN), CHMP4B Smartpool
(L-018075-01-0005, Horizon Discovery), Stx17 Smartpool (M-
020965-01-0005, Horizon Discovery), ATG7 Smartpool (L-020112-
00-0005, Horizon Discovery), Ykt6 siRNA directed against sense
5′-ATTCATTGTCAGCAATGACCACACC-3′ (Matsui et al., 2018),
Vps4A siRNA directed against sense 5′-CTGTGGTTTGCATGTC-
GGA-3′ (Mejlvang etal., 2018), Vps4B siRNA directed against sense
5′-CCAAAGAAGCACTGAAAGA-3′ (Mejlvang et al., 2018). The
2xFYVE-mCherry construct was ordered from AddGene (deposited
by Harald Stenmark, 140050). The EEA1-mCherry construct was or-
dered from AddGene (deposited by Yusuke Ohba, #174452). The
EEA1-mCherry fragment from this vector was cloned into a pmini-
Tol2 expression vector (original deposited on AddGene by Stephen
Ekker, #31829, we obtained a modied backbone with EF1α pro-
motor from Jooske Monster, CMM, UMCU) using restriction-liga-
tion. Vector with Transposase from Oryzias latipes was obtained
from Jooske Monster, CMM, UMCU.
Cell lines
HeLa parental cells were obtained from DSMZ (ACC 57), Vps11,
Vps18, Vps39 and Vps41 knock-outs were generated as described
previously (van der Welle etal., 2021). The A549 HOPS KO cell lines
were obtained using gRNA sequence 5′-AGTGGGGGATGCCCAC-
GCTA-3′ for Vps18 and gRNA sequence 5′-AAGTATTTCAGT-
TACCCCAT-3′ for Vps41. For HOPS KO in HT1080 cell lines, we
used gRNA sequence 5′-GCGAGTTCTCGTACTCATCC-3′ for Vps18
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 19
and gRNA sequence 5′-GCCAGACAGTCGATTTGCAG-3′ for
Vps39. The lines were validated by Western blot (Supplementary
Figure S4, A and B), or for Vps39 by genomic DNA isolation and
sequencing (Supplementary Figure S4C), which revealed a single
base deletion at the expected cut site for both alleles.
EEA1-mCherry−expressing cell lines were obtained by cloning
EEA1-mCherry into a minitol vector designed for stable expression
under an EF-1α promotor. HeLa WT, Vps18, and Vps39 KO lines
were transfected with this vector and a vector expressing a trans-
posase. After 48 h, puromycin was added to the medium at 1 µg/ml
for selection of cells that had the minitol vector stably integrated.
Cell culture and transfection
HeLa, A549, and HT1080 cells and CRISPR/Cas9 KO lines were cul-
tured in DMEM supplemented with 20% FCS and penicillin and
streptomycin. They were incubated in 37°C, 5% CO2 incubators,
passaged twice per week on average, and kept in culture until pas-
sage 20. For transfection of cells using plasmids, cells were incu-
bated with effectene and plasmid mix ON and given normal medium
4 h before xation or imaging. For knock-down of cells, we used Hi-
Perfect and siRNAs at 20 nM end concentration. The HiPerfect mix
was added to culture medium. After 72 h, cells were treated again
with siRNAs for another 72 h before use in experiments. The ef-
ciency of the knock-down was validated by QPCR and ≥80% reduc-
tion was obtained for all experiments used in this study.
Dextran uptake assay
Cells were incubated with SiRlysosome (SC012, Spirochrome),
1:1000 in normal DMEM medium for 60 min. Then dextran-AF488
was added to the SiRlysosome-medium at nal concentration 0.2
mg/ml, incubated for 2 h, washed ve times with fresh, 37°C DMEM,
and xed in 4% FA. If needed, additional IF staining was performed
using the protocol described below, otherwise the sample was
washed three times in PBS, once in H2O and mounted using Pro-
Long Dapi Diamond (P36962, Thermo Fisher Scientic). Slides were
imaged within 3 d after xation.
Western blotting
For analysis of whole-cell lysates by Western blot, cells were cul-
tured in standard cell culture plates, placed on ice, washed with
ice-cold PBS, and scraped and lysed in 1% CHAPS lysis buffer. The
samples were spun down at ≥10,000 × g for 10 min at 4°C to remove
Name Host Catalog number Company Dilutions
LAMP1 Mse 555798 BD Pharmingen 1:500 (IF)
cathepsin D Gt AF1014 R&D Systems 1:500 (IF), 1:1000 (WB)
cathepsin B Gt AF953 R&D Systems 1:1000 (WB)
Calnexin Rb Ab22595 Abcam 1:1000 (WB)
Rab5 Mse 610725 BD Biosciences 1:250 (IF)
Rab7 Rb 9367 Cell Signaling Technology 1:250 (IF)
Vps11 Rb ab170869 Abcam 1:500 (WB)
Vps18 Rb ab178416 Abcam 1:500 (WB)
Vps41 Mse sc-377118 Santa Cruz Biotechnology 1:1000 (WB)
CD63 Mse H5C6 DSHB 1:500 (IF)
EEA1 Mse 610457 BD Transduction 1:250 (IF), 1:150 (CLEM)
EEA1 Rb C45B10 Cell Signaling Technology 1:250 (IF)
Vps35 Gt Ab10099 Abcam 1:500 (IF)
CI-MPR Rb Kind gift from A. Hille/K. von Figura 1:300 (IF), 1:50 (EM)
CI-MPR Rb Kind gift from S. Kornfeld 1:1000 (WB)
SNX1 Rb Kind gift from P. Cullen 1:150 (EM)
SNX1 Mse 611482 BD Biosciences 1:250 (IF)
TGN46 Shp AHP500 Bio-Rad (Serotec) 1:500 (IF)
GM130 Mse Kind gift from E. Sztul 1:250 (IF)
p62 Mse 610833 BD Biosciences 1:1000 (WB)
p62 GnP GP62-C Sanbio 1:100 (EM)
LC3 Mse CTB-LC3-2-IC Cosmo Bio 1:10 (EM)
AF488 anti-mouse Dk A21202 Life Technologies 1:250 (IF)
AF568 anti-rabbit Dk A10042 Life Technologies 1:250 (IF)
AF647 anti-goat Dk A21447 Life Technologies 1:250 (IF)
AF488 anti-goat Dk A11055 Life Technologies 1:250 (IF)
AF555 plus anti-mouse Dk A32773 Thermo Fisher Scientic 1:250 (IF)
Anti-mouse Rb 610-4120 Rockland 1:300 (EM)
TABLE 1: The following antibodies were used in this manuscript.
20 | J. van der Beek et al. Molecular Biology of the Cell
cellular debris and equalized using a Bradford protein assay (Bio-
Rad). SDS sample buffer was added, samples were run on precast
4−20% gradient gels (Bio-Rad) and transferred to Mini PVDF mem-
branes (Bio-Rad) using the Trans-Blot TurboTransfer system (Bio-
Rad). The membranes were blocked in Odyssey PBS Blocking buffer
(LI-COR) at room temperature for 1 h and then incubated with pri-
mary antibodies in 0.1% TBST overnight at 4°C. Membranes were
washed in 0.1% TBST and incubated with secondary antibodies
for 1 h at room temperature before being washed in 0.1% TBST,
followed by PBS and then H2O. Membranes were scanned on an
Amersham Typhoon Laser scanner (GE Healthcare).
For analysis of culture medium, cells were cultured in 15-cm
dishes and incubated with serum-free medium overnight. The me-
dium was collected, spun at 100,000 × g to remove any debris or
particles, and the supernatant was precipitated by adding 10%
trichloroacetic acid (TCA). The mixture was placed on ice for 30 min
and then spun down at 10,000 × g at 4°C for 15 min. The superna-
tant was aspirated and washed with acetone at 4°C. The dry pellet
was then resuspended in SDS buffer and Western blotting was per-
formed as described above. For loading control, the corresponding
cells from the 15-cm dish were processed as described above, with
the exception that they were not equalized. The protein concentra-
tion in these samples therefore reects the number of cells in the
dish.
Immunofluorescence staining
Cells were prepared for IF staining by seeding on glass coverslips
and xation with 4% FA for 15−20 min. The coverslips were washed
with PBS three times, permeabilized in 0.1% TritonX-100 for 10 min
at room temperature, and blocked in 1% BSA in PBS for 10 min at
room temperature. Coverslips were incubated with primary anti-
bodies diluted in 1% BSA for 1 h at room temperature followed by
three PBS washes and incubation with secondary antibodies for
30 min at room temperature. Coverslips were then washed three
times with PBS, once in H2O, and mounted on glass slides using
ProLong DAPI Diamond (P36962, Thermo Fisher Scientic).
Slides were imaged at room temperature using a Leica Thunder
microscope with 100×, 1.47 NA TIRF oil objective or a DeltaVision
with 100×, 1.40 NA oil objective. For confocal imaging, slides were
imaged on a Zeiss LSM 700 with 63×, 1.40 NA oil objective at room
temperature.
pH measurements
The pH measurements were performed as described by Canton and
Grinstein (Canton and Grinstein, 2015). To obtain a calibration data-
set, HeLa WT cells were incubated with 0.2 mg/ml Dextran-uores-
cein in normal medium for 3 h, washed four times with culture me-
dium, and incubated in a pH-buffered solution containing 140 mM
KCl, 1 mM MgCl2, 1 mM CaCl, 5 mM glucose, and 25 mM of ace-
tate-acetic acid, MES or HEPES buffer, depending on the desired
pH. Immediately before imaging, Nigericin was added to an end
concentration of 10 µM to equalize endo-lysosomal pH to the buff-
ered pH. Cells were then imaged with 440 and 480 excitation and a
uorescein emission lter on a Leica Thunder microscope with 100×,
1.40 NA oil objective and 37°C, 5% CO2 live-cell imaging chamber.
Emission ratios of uorescein 440/480 excitation channels were ob-
tained for seven samples buffered in a range from pH 4 to pH 8. A
sigmoid curve was tted to describe the relation of uorescein ratio
and pH, which was used to calculate pH from the live-cell measure-
ments subsequently taken (Supplementary Figure S7). We bench-
marked our calibration by live-cell imaging and measuring the aver-
age pH of 3-h uptake Dextran-uorescein−positive compartments in
HeLa WT cells, which returned an expected acidied pH of 5.1
(Supplementary Figure S7, C and D). Continuing, HeLa WT, Vps18,
and Vps39 KO cells stably expressing EEA1-mCherry were incu-
bated with 0.2 mg/ml Dextran-uorescein for 3 h, washed four times
and imaged in phenol-red free, otherwise normal culture medium.
Samples were then imaged on a Leica Thunder microscope. The
emission ratios of uorescein 440/480 excitation channels were ob-
tained on a per-compartment basis for >10 cells. Analysis of com-
partment intensity and ratios was performed in FIJI using a custom
macro. The formula from the sigmoid curve tted to the calibration
samples was used to calculate pH values from the emission ratios of
uorescein 440/480 excitation.
Image analysis
Light microscopy images were analyzed using FIJI (Schindelin etal.,
2012). The ComDet Plug-In (Eugene Katrukha, Cell Biology, Utrecht
University) was used for spot detection, spot size quantication,
spot intensity measurements and colocalization analysis. EEA1 and
CI-MPR colocalization was measured as the percentage of overlap
between thresholded signal masks of the two channels. All mea-
surements were performed per cell in a custom cell-segmenting
macro.
For quantication of CLEM data, organelles were selected based
on the presence of uorescent EEA1 signal and categorized based
on morphological characteristics described below.
Morphological analysis of the HPF samples was done by random
selection of ∼200 endocytic organelles per condition. Multiple char-
acteristics were measured for each organelle, which were then cat-
egorized on the basis of morphological characteristics described
below.
For quantication of p62-positive content in endosomal organ-
elles (Figure 9D), ∼50 BSA5-positive organelles were imaged for
each condition and scored for the number of gold particles repre-
senting p62. Organelles with 0 particles were regarded as negative,
organelles with 1 particle were excluded, and organelles with >1
particle were considered p62-positive.
EM of resin-embedded, high-pressure frozen cells
Cells were seeded on sapphire disks for 24 h and incubated with
BSA5 for 3 h before HPF. 10 min before HPF, cells were washed with
full DMEM. The sapphire disks and cells were transferred into sam-
ple holders and inserted into a Leica EM ICE (Leica Microsystems)
and frozen. After freezing, the samples were stored in LN2. Samples
were freeze substituted with chemical xative and postxed in 2%
OsO4. Cells were further processed in EPON in increasing concen-
trations and nally embedded in 100% EPON, polymerized for 3 d
at 60°C.
Resin embedded cells were prepared for thin sectioning by re-
moval of the sapphire disk and trimming of the stub to a rectangle
of ∼0.5 by 1 µm. 60−70 nm Sections were made on a Leica Ultracut
S (Leica microsystems) using a DiATOME Ultra Diamond Knife 45°.
Sections were deposited on Formvar- and carbon-coated copper
grids and poststained using uranyl and lead citrate in a Leica EM
AC20 (Leica microsystems). Samples were imaged on a Tecnai T12
(FEI Tecnai) transmission electron microscope (TEM) using SerialEM
software (Mastronarde, 2018). Image tileset stitching was done with
Etomo EM processing software (Mastronarde and Held, 2017).
On-section CLEM
A detailed protocol for this procedure is available in van der Beek
etal. (van der Beek etal., 2022, 2023) Briey, cells were xed with
4% FA overnight, washed, and scraped before being pelleted and
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 21
embedded in 12% (wt/vol) gelatin. Embedded cell pellets were cut
into smaller (0.5 mm3) blocks, mounted on aluminum pins and snap
frozen in LN2.
Frozen samples were trimmed and sectioned to 90-100 nm on a
Leica UC7 Cryo Microtome using a DiATOME Cryo Diamond Knife
45°. The sections were picked up using a 1:1 mixture of 2.3M su-
crose and 1.8% methylcellulose and deposited on Formvar- and
carbon-coated copper grids. Grids were incubated in PBS at 37°C
for 30 min, washed with PBS 0.15% glycine, blocked in PBS with
BSA-c and sh-skin gelatin, and incubated with primary antibodies
for 1 h at room temperature. The grids were then washed and incu-
bated with uorescent secondary antibodies for 30 min at room
temperature. After further washes, grids were sandwiched between
clean slideglasses and coverslips in 50% Glycerol and imaged on a
Leica Thunder wideeld microscope using a 100×, 1.47 NA TIRF
objective at room temperature. After acquisition of an image tileset
of the sections, the grids were retrieved from the slideglass, washed,
and stained using Uranyl Acetate. The grids were then imaged in a
Tecnai T12 TEM (FEI Tecnai) using SerialEM, selecting areas based
on uorescence microscopy. Image tilesets of the selected areas
were generated at 42,000×, stitched together in Etomo postpro-
cessing software and correlated with the uorescence data (Paul-
Gilloteaux etal., 2017).
IF on sections
Samples and sections were obtained as under “On-section CLEM”
above, except that 150-nm sections were obtained and they were
deposited on 10-mm coverslips. They were processed in the same
manner as CLEM samples up to secondary antibody incubation, af-
ter which they were washed and mounted on slideglasses using Pro-
Long DAPI Diamond (P36962, Thermo Fisher Scientic). They were
imaged as described under “Immunouorescence staining”.
Immuno-EM
Immunogold labeling of thawed cryosections was performed as de-
veloped and optimized in our lab, for details see (Slot and Geuze,
2007; De Mazière etal., 2022). The procedure follows the protocol
for CLEM up to and including sectioning. After sectioning, grids
were placed on PBS at 37°C for 30 min, washed with PBS 0.15%
glycine, blocked in PBS with BSA-c and sh-skin gelatin, and labeled
with primary antibodies for 1 h at room temperature. Grids were
washed, incubated with a bridging rabbit anti-mouse antibody for
20 min at room temperature, washed again, and incubated with Pro-
tein A coupled to 10 or 15 nm gold (PAG) particles for 20 min at
room temperature. In case of double labeling, the grids were xed
in 1% GA for 5 min after the rst labeling, washed, and then the
labeling was repeated with a second primary antibody. After PAG
labeling, grids were washed extensively in H2O before staining with
Uranyl Acetate. Immuno-EM samples were imaged on a Tecnai T12
TEM or Tecnai T20 (FEI Tecnai) using SerialEM or Radius image ac-
quisition software.
Morphological definitions of the endo-lysosomal system
The EM morphology of the endo-lysosomal system has been stud-
ied for many decades. Immunogold labeling and functional assays
have elucidated key structure−function relationships that are gener-
ally applied to categorize the different endo-lysosomal subtypes
(Raiborg et al., 2002; Mari et al., 2008; Eskelinen et al., 2011;
Klumperman and Raposo, 2014; Fermie et al., 2018; Davis et al.,
2021; Barral etal., 2022; van der Beek etal., 2022; ). Based on these
collective ndings, we used the following denitions for morpho-
logical denition of distinct endo-lysosomal organelles. Although
these denitions are not absolute, they allow us to interpret ultra-
structural ndings and changes in a nonarbitrary way.
Early endosomes: largely electron-lucent organelles with <6 ILVs,
frequently displaying at, bi-layered clathrin coats on their limiting
membrane and forming tubular extensions. Late endosomes:
rounded shape, lumen-containing medium electron-dense content
and >5 ILVs. Lysosomes: vacuoles of variable size and shape, amor-
phous content ranging from electron lucent to highly electron-
dense, ILVs, and onion-like concentric rings of membranes. HOPS
bodies displayed both early and late endosomal features as clathrin
coats, associated tubules, ILVs, membranes, and amorphous and
regularly dense granules, sometimes surrounded by a membrane,
indicative of autophagic cargos. Some of these features are also
displayed by late endosomes and (auto)lysosomes in WT cells,
which is why our control cells contained low, but nonzero numbers
of “HOPS bodies”.
ACKNOWLEDGMENTS
We thank our colleagues from the Center for Molecular Medicine
and especially Catherine Rabouille of the Hubrecht Laboratory for
fruitful discussions and feedback. We thank Matteo for support and
advice on some of the experiments. We warmly thank Bas van Zui-
jlen, Susan Zwakenberg, and Fried Zwartkruis for help with generat-
ing knock-out cell lines. N.L. is supported by a ZonMw TOP grant
(40-00812-98-16006 to J.K.). P.S. is supported by a DFG grant
(FOR2625 to J.K. as part of the Research Consortium). The EM
infrastructure used in this work is part of the Netherlands Electron
Microscopy Infrastructure (NEMI), a research program National
Roadmap for Large-Scale Research Infrastructure, which is nanced
by the Dutch Research Council to J.K. (project number 184.034.014).
REFERENCES
Anand VC, Daboussi L, Lorenz TC, Payne GS (2009). Genome-wide
analysis of AP-3–dependent protein transport in yeast. Mol Biol Cell 20,
1592–1604.
Anderson J, Walker G, Pu J (2022). BORC-ARL8-HOPS ensemble is required
for lysosomal cholesterol egress through NPC2. Mol Biol Cell 33, ar81.
Baker RW, Jeffrey PD, Zick M, Phillips BP, Wickner WT, Hughson FM (2015).
A direct role for the Sec1/Munc18-family protein Vps33 as a template
for SNARE assembly. Science 349, 1111–1114.
Balderhaar HJK, Ungermann C (2013). CORVET and HOPS tethering com-
plexes - coordinators of endosome and lysosome fusion. J Cell Sci 126,
1307–1316.
Ballabio A, Bonifacino JS (2020). Lysosomes as dynamic regulators of cell
and organismal homeostasis. Nat Rev Mol Cell Biol 21, 101–118.
Barral DC, Staiano L, Guimas Almeida C, Cutler DF, Eden ER, Futter CE,
Galione A, Marques ARA, Medina DL, Napolitano G, etal. (2022).
Current methods to analyze lysosome morphology, positioning, motility
and function. Trafc 23, 238.
Bas L, Papinski D, Licheva M, Torggler R, Rohringer S, Schuschnig M, Kraft C
(2018). Reconstitution reveals Ykt6 as the autophagosomal SNARE in
autophagosome-vacuole fusion. J Cell Biol 217, 3656–3669.
Bowman EJ, Siebers A, Altendorf K (1988). Balomycins; A class of inhibi-
tors of membrane ATPases from microorganisms, animal cells, and plant
cells. Proc Natl Acad Sci USA 85, 7972–7976.
Braulke T, Bonifacino JS (2009). Sorting of lysosomal proteins. Biochim
Biophys Acta 1793, 605–614.
Brocker C, Kuhlee A, Gatsogiannis C, Kleine Balderhaar HJ, Honscher C,
Engelbrecht-Vandre S, Ungermann C, Raunser S (2012). Molecular
architecture of the multisubunit homotypic fusion and vacuole protein sort-
ing (HOPS) tethering complex. Proc Natl Acad Sci USA 109, 1991–1996.
Brown WJ, Constantinescu E, Farquhar MG (1984). Redistribution
of mannose-6-phosphate receptors induced by tunicamycin and
chloroquine. J Cell Biol 99, 320–326.
Brown WJ, Farquhar MG (1984). The mannose-6-phosphate receptor for lyso-
somal enzymes is concentrated in cis Golgi cisternae. Cell 36, 295–307.
Brown WJ, Goodhouse J, Farquhar MG (1986). Mannose-6-phosphate
receptors for lysosomal enzymes cycle between the Golgi complex and
endosomes. J Cell Biol 103, 1235–1247.
22 | J. van der Beek et al. Molecular Biology of the Cell
Burd C, Cullen PJ (2014). Retromer: a master conductor of endosome
sorting. Cold Spring Harb Perspect Biol 6, a016774 .
Canton J, Grinstein S (2015). Measuring lysosomal pH by uorescence
microscopy. Methods Cell Biol 126, 85–99.
Chou HT, Dukovski D, Chambers MG, Reinisch KM, Walz T (2016). CATCHR,
HOPS and CORVET tethering complexes share a similar architecture.
Nat Struct Mol Biol 23, 761–763.
Collins KM, Thorngren NL, Fratti RA, Wickner WT (2005). Sec17p and
HOPS, in distinct SNARE complexes, mediate SNARE complex
disruption or assembly for fusion. EMBO J 24, 1775–1786.
Collins KM, Wickner WT (2007). trans-SNARE complex assembly and yeast
vacuole membrane fusion. Proc Natl Acad Sci USA 104, 8755–8760.
Cowles CR, Snyder WB, Burd CG, Emr SD (1997). Novel Golgi to vacuole
delivery pathway in yeast: Identication of a sorting determinant and
required transport component. EMBO J 16, 2769–2782.
D’Agostino M, Risselada HJ, Lürick A, Ungermann C, Mayer A (2017). A
tethering complex drives the terminal stage of SNARE-dependent
membrane fusion. Nature 551, 634–638.
Davis LJ, Bright NA, Edgar JR, Parkinson MDJ, Wartosch L, Mantell J,
Peden AA, Luzio JP (2021). Organelle tethering, pore formation and
SNARE compensation in the late endocytic pathway. J Cell Sci 134,
jcs255463. doi:10.1242/jcs.255463.
Delevoye C, Marks MS, Raposo G (2019). Lysosome-related organelles as
functional adaptations of the endolysosomal system. Curr Opin Cell Biol
59, 147–158.
De Mazière A, van der Beek J, van Dijk S, de Heus C, Reggiori F, Koike
M, Klumperman J (2022). An optimized protocol for immuno-electron
microscopy of endogenous LC3. Autophagy 18, 3004–3022.
Eskelinen EL, Reggiori F, Baba M, Kovács AL, Seglen PO (2011). Seeing is
believing: The impact of electron microscopy on autophagy research.
Autophagy 7, 935–956.
Evans AJ, Daly JL, Anuar ANK, Simonetti B, Cullen PJ (2020). Acute
inactivation of retromer and ESCPE-1 leads to time-resolved defects in
endosomal cargo sorting. J Cell Sci 133, jcs246033.
Fermie J, Liv N, ten Brink C, van Donselaar EG, Müller WH, Schieber NL,
Schwab Y, Gerritsen HC, Klumperman J (2018). Single organelle dynam-
ics linked to 3D structure by correlative live-cell imaging and 3D electron
microscopy. Trafc 19, 354–369.
Ganesan D, Cai Q (2021). Understanding amphisomes. Biochem J 478,
1959–1976.
Gillingham AK, Bertram J, Begum F, Munro S (2019). In vivo identication
of GTPase interactors by mitochondrial relocalization and proximity
biotinylation. Elife 8, e45916.
Gillingham AK, Sinka R, Torres IL, Lilley KS, Munro S (2014). Toward a com-
prehensive map of the effectors of rab GTPases. Dev Cell 31, 358–373.
Gillooly DJ, Raiborg C, Stenmark H (2003). Phosphatidylinositol 3-phos-
phate is found in microdomains of early endosomes. Histochem Cell
Biol 120, 445–453.
Graham SC, Wartosch L, Gray SR, Scoureld EJ, Deane JE, Luzio JP, Owen
DJ (2013). Structural basis of Vps33A recruitment to the human HOPS
complex by Vps16. Proc Natl Acad Sci USA 110, 13345–13350.
Hämälistö S, Jäättelä M (2016). Lysosomes in cancer - living on the edge
(of the cell). Curr Opin Cell Biol 39, 69–76.
Hämälistö S, Stahl JL, Favaro E, Yang Q, Liu B, Christoffersen L, Loos B,
Guasch Boldú C, Joyce JA, Reinheckel T, etal. (2020). Spatially and
temporally dened lysosomal leakage facilitates mitotic chromosome
segregation. Nat Commun 11, 229.
Herrmann E, Langemeyer L, Auffarth K, Ungermann C, Kümmel D (2023).
Targeting of the Mon1-Ccz1 Rab guanine nucleotide exchange factor to
distinct organelles by a synergistic protein and lipid code. J Biol Chem
299, 102915.
Hille-Rehfeld A (1995). Mannose 6-phosphate receptors in sorting and
transport of lysosomal enzymes. Biochim Biophys Acta 1241, 177–194.
Homewood CA, Warhurst DC, Peters W, Baggaley VC (1972). Lysosomes,
pH and the anti-malarial action of chloroquine. Nature 235, 50–52.
Hu Y, Reggiori F (2022). Molecular regulation of autophagosome formation.
Biochem Soc Trans 50, 55–69.
Huotari J, Helenius A (2011). Endosome maturation. EMBO J 30, 3481–
3500.
Jiang P, Nishimura T, Sakamaki Y, Itakura E, Hatta T, Natsume T, Mizushima
N (2014). The HOPS complex mediates autophagosome-lysosome
fusion through interaction with syntaxin 17. Mol Biol Cell 25, 1327–1337.
Jongsma ML, Bakker J, Cabukusta B, Liv N, van Elsland D, Fermie J,
Akkermans JL, Kuijl C, van der Zanden SY, Janssen L, etal. (2020).
SKIP-HOPS recruits TBC1D15 for a Rab7-to-Arl8b identity switch to
control late endosome transport. EMBO J 39, e102301.
Jonker CTH, Galmes R, Veenendaal T, Ten Brink C, van der Welle REN, Liv N,
de Rooij J, Peden AA, van der Sluijs P, Margadant C, Klumperman J (2018).
Vps3 and Vps8 control integrin trafcking from early to recycling endo-
somes and regulate integrin-dependent functions. Nat Commun 9, 792.
Kajiho H, Kajiho Y, Frittoli E, Confalonieri S, Bertalot G, Viale G, Di Fiore
PP, Oldani A, Garre M, Beznoussenko GV, etal. (2016). RAB2A controls
MT1-MMP endocytic and E-cadherin polarized Golgi trafcking to pro-
mote invasive breast cancer programs. EMBO Rep 17, 1061–1080.
Khatter D, Raina VB, Dwivedi D, Sindhwani A, Bahl S, Sharma M (2015).
The small GTPase Arl8b regulates assembly of the mammalian HOPS
complex on lysosomes. J Cell Sci 128, 1746–1761.
Kim BY, Krämer H, Yamamoto A, Kominami E, Kohsaka S, Akazawa C (2001).
Molecular characterization of mammalian homologues of class C Vps
proteins that interact with syntaxin-7. J Biol Chem 276, 29393–29402.
Klumperman J, Hille A, Veenendaal T, Oorschot V, Stoorvogel W, von Figura
K, Geuze HJ (1993). Differences in the endosomal distributions of the
two mannose 6-phosphate receptors. J Cell Biol 121, 997–1010.
Klumperman J, Raposo G (2014). The complex ultrastructure of the endoly-
sosomal system. Cold Spring Harb Perspect Biol 6, a016857.
Kumar AV, Mills J, Lapierre LR (2022). Selective autophagy receptor p62/
SQSTM1, a pivotal player in stress and aging. Front Cell Dev Biol 10,
793328.
Langemeyer L, Borchers A-C, Herrmann E, Füllbrunn N, Han Y, Perz A,
Auffarth K, Kümmel D, Ungermann C (2020). A conserved and regulated
mechanism drives endosomal Rab transition. eLife 9, e56090.
Laurent-Matha V, Derocq D, Prébois C, Katunuma N, Liaudet-Coopman E
(2006). Processing of human cathepsin D is independent of its catalytic
function and auto-activation: involvement of cathepsins L and B.
J Biochem 139, 363–371.
Lie PPY, Nixon RA (2019). Lysosome trafcking and signaling in health and
neurodegenerative diseases. Neurobiol Dis 122, 94–105.
Lin X, Yang T, Wang S, Wang Z, Yun Y, Sun L, Zhou Y, Xu X, Akazawa C,
Hong W, Wang T (2014). RILP interacts with HOPS complex via VPS41
subunit to regulate endocytic trafcking. Sci Rep 4, 7282.
Liv N, Fermie J, Ten Brink CBM, de Heus C, Klumperman J (2023). Functional
characterization of endo-lysosomal compartments by correlative live-cell
and volume electron microscopy. Methods Cell Biol 177, 301–326.
Llinares E, Barry AO, André B (2015). The AP-3 adaptor complex mediates
sorting of yeast and mammalian PQ-loop-family basic amino acid trans-
porters to the vacuolar/lysosomal membrane. Sci Rep 5, 16665.
Lőrincz P, Juhász G (2020). Autophagosome-lysosome fusion. J Mol Biol
432, 2462–2482.
Lőrincz P, Tóth S, Benkő P, Lakatos Z, Boda A, Glatz G, Zobel M, Bisi S,
Hegedűs K, Takáts S, etal. (2017). Rab2 promotes autophagic and
endocytic lysosomal degradation. J Cell Biol 216, 1937–1947.
Lürick A, Kuhlee A, Bröcker C, Kümmel D, Raunser S, Ungermann C (2015).
The Habc domain of the SNARE Vam3 interacts with the HOPS tethering
complex to facilitate vacuole fusion. J Biol Chem 290, 5405.
Luzio JP, Parkinson MDJ, Gray SR, Bright NA (2009). The delivery of
endocytosed cargo to lysosomes. Biochem Soc Trans 37, 1019–1021.
Mari M, Bujny MV, Zeuschner D, Geerts WJCC, Grifth J, Petersen CM,
Cullen PJ, Klumperman J, Geuze HJ (2008). SNX1 denes an early
endosomal recycling exit for sortilin and mannose 6-phosphate
receptors. Trafc 9, 380–393.
Marques ARAA, Saftig P (2019). Lysosomal storage disorders - challenges,
concepts and avenues for therapy: beyond rare diseases. J Cell Sci 132,
jcs221739.
Mastronarde DN (2018). Advanced data acquisition from electron
microscopes with SerialEM. Microsc Microanal 24, 864–865.
Mastronarde DN, Held SR (2017). Automated tilt series alignment and
tomographic reconstruction in IMOD. J Struct Biol 197, 102–113.
Matsui T, Jiang P, Nakano S, Sakamaki Y, Yamamoto H, Mizushima N (2018).
Autophagosomal YKT6 is required for fusion with lysosomes indepen-
dently of syntaxin 17. J Cell Biol 217, 2633–2645.
McEwan DG, Popovic D, Gubas A, Terawaki S, Suzuki H, Stadel D, Coxon
FP, MirandadeStegmann D, Bhogaraju S, Maddi K, etal. (2015).
PLEKHM1 regulates autophagosome-lysosome fusion through HOPS
complex and LC3/GABARAP proteins. Mol Cell 57, 39–54.
Mejlvang J, Olsvik H, Svenning S, Bruun J-A, Abudu YP, Larsen KB, Brech A,
Hansen TE, Brenne H, Hansen T, etal. (2018). Starvation induces rapid
degradation of selective autophagy receptors by endosomal microau-
tophagy. J Cell Biol 217, 3640–3655.
Miao G, Zhao H, Li Y, Ji M, Chen Y, Shi Y, Bi Y, Wang P, Zhang H (2021).
ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-
mediated assembly of the SNARE complex required for autolysosome
formation. Dev Cell 56, 427–442.e5.
Volume 35 March 1, 2024 HOPS KO disrupts endosomal organization | 23
Mizushima N (2020). The ATG conjugation systems in autophagy. Curr Opin
Cell Biol 63, 1–10.
Monfrini E, Cogiamanian F, Salani S, Straniero L, Fagiolari G, Garbellini
M, Carsana E, Borellini L, Biella F, Moggio M, etal. (2021a). A novel
homozygous VPS11 variant may cause generalized dystonia. Ann Neurol
89, 834–839.
Monfrini E, Zech M, Steel D, Kurian MA, Winkelmann J, Di Fonzo A
(2021b). HOPS-associated neurological disorders (HOPSANDs): linking
endolysosomal dysfunction to the pathogenesis of dystonia. Brain 144,
2610–2615.
Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG,
Campbell PL, McCluskey J, Yeo JP, Tock EPC, Toh BH (1995). EEA1,
an early endosome-associated protein. EEA1 is a conserved α- helical
peripheral membrane protein anked by cysteine “ngers” and contains
a calmodulin-binding IQ motif. J Biol Chem 270, 13503–13511.
Murray DH, Jahnel M, Lauer J, Avellaneda MJ, Brouilly N, Cezanne A,
Morales-Navarrete H, Perini ED, Ferguson C, Lupas AN, etal. (2016).
An endosomal tether undergoes an entropic collapse to bring vesicles
together. Nature 537, 107–111.
Olson LJ, Misra SK, Ishihara M, Battaile KP, Grant OC, Sood A, Woods RJ,
Kim JJP, Tiemeyer M, Ren G, etal. (2020). Allosteric regulation of
lysosomal enzyme recognition by the cation-independent mannose
6-phosphate receptor. Commun Biol 3, 498.
Paul-Gilloteaux P, Heiligenstein X, Belle M, Domart MC, Larijani B, Collinson
L, Raposo G, Salamero J (2017). EC-CLEM: Flexible multidimensional
registration software for correlative microscopies. Nat Methods 14,
102–103.
Peng C, Ye J, Yan S, Kong S, Shen Y, Li C, Li Q, Zheng Y, Deng K, Xu T, Tao
W (2012). Ablation of vacuole protein sorting 18 (Vps18) gene leads
to neurodegeneration and impaired neuronal migration by disrupting
multiple vesicle transport pathways to lysosomes. J Biol Chem 287,
32861–32873.
Peplowska K, Markgraf DF, Ostrowicz CW, Bange G, Ungermann C (2007).
The CORVET tethering complex interacts with the yeast Rab5 homolog
Vps21 and is involved in endo-lysosomal biogenesis. Dev Cell 12,
739–750.
Perini ED, Schaefer R, Stöter M, Kalaidzidis Y, Zerial M (2014). Mammalian
CORVET is required for fusion and conversion of distinct early endo-
some subpopulations. Trafc 15, 1366–1389.
Pols MS, ten Brink C, Gosavi P, Oorschot V, Klumperman J (2013a). The
HOPS proteins hVps41 and hVps39 are required for homotypic and
heterotypic late endosome fusion. Trafc 14, 219–232.
Pols MS, van Meel E, Oorschot V, ten Brink C, Fukuda M, Swetha MG,
Mayor S, Klumperman J (2013b). hVps41 and VAMP7 function in
direct TGN to late endosome transport of lysosomal membrane
proteins. Nat Commun 4, 1361.
Poteryaev D, Datta S, Ackema K, Zerial M, Spang A (2010). Identication
of the switch in early-to-late endosome transition. Cell 141, 497–508.
Pu J, Guardia CM, Keren-Kaplan T, Bonifacino JS (2016). Mechanisms and
functions of lysosome positioning. J Cell Sci 129, 4329–4339.
Raiborg C, Bache KG, Gillooly DJ, Madshus IH, Stang E, Stenmark H (2002).
Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of
early endosomes. Nat Cell Biol 4, 394–398.
Reggiori F, Ungermann C (2017). Autophagosome maturation and fusion.
J Mol Biol 429, 486–496.
Reitman ML, Varki A, Kornfeld S (1981). Fibroblasts from patients with
I-cell disease and pseudo-Hurler polydystrophy are decient in uridine
5’-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosami-
nylphosphotransferase activity. J Clin Invest 67, 1574–1579.
Rieder SE, Emr SD (1997). A novel RING fringer protein complex essential
for a late step in protein transport to the yeast vacuole. Mol Biol Cell 8,
2307–2327.
Rink J, Ghigo E, Kalaidzidis Y, Zerial M (2005). Rab conversion as a
mechanism of progression from early to late endosomes. Cell 122,
735–749.
Saftig P, Klumperman J (2009). Lysosome biogenesis and lysosomal mem-
brane proteins: Trafcking meets function. Nat Rev Mol Cell Biol 10,
623–635.
Sato TK, Rehling P, Peterson MR, Emr SD (2000). Class C Vps protein
complex regulates vacuolar SNARE pairing and is required for vesicle
docking/fusion. Mol Cell 6, 661–671.
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch
T, Preibisch S, Rueden C, Saalfeld S, Schmid B, etal. (2012). Fiji: An
open-source platform for biological-image analysis. Nat Methods 9,
676–682.
Schleinitz A, Pöttgen L-A, Keren-Kaplan T, Pu J, Saftig P, Bonifacino JS,
Haas A, Jeschke A (2023). Consecutive functions of small GTPases guide
HOPS-mediated tethering of late endosomes and lysosomes. Cell Rep
42, 111969.
Schwartz ML, Nickerson DP, Lobingier BT, Plemel RL, Duan M, Angers CG,
Zick M, Merz AJ (2017). Sec17 (α-SNAP) and an SM-tethering complex
regulate the outcome of SNARE zippering in vitro and in vivo. eLife 6,
e27396.
Seals DF, Eitzen G, Margolis N, Wickner WT, Price A (2000). A Ypt/Rab
effector complex containing the Sec1 homolog Vps33p is required for
homotypic vacuole fusion. Proc Natl Acad Sci 97, 9402–9407.
Seaman MNJ (2004). Cargo-selective endosomal sorting for retrieval to the
Golgi requires retromer. J Cell Biol 165, 111–122.
Shvarev D, Schoppe J, König C, Perz A, Füllbrunn N, Kiontke S, Lange-
meyer L, Januliene D, Schnelle K, Kümmel D, etal. (2022). Structure of
the HOPS tethering complex, a lysosomal membrane fusion machinery.
eLife 11, e80901.
Simonetti B, Daly JL, Cullen PJ (2023). Out of the ESCPE room: Emerging
roles of endosomal SNX-BARs in receptor transport and host-pathogen
interaction. Trafc 24, 234–250.
Slot JW, Geuze HJ (2007). Cryosectioning and immunolabeling. Nat Protoc
2, 2480–2491.
Song H, Orr AS, Lee M, Harner ME, Wickner WT (2020). HOPS recognizes
each SNARE, assembling ternary trans-complexes for rapid fusion upon
engagement with the 4th SNARE. eLife 9, e53559.
Sriram V, Krishnan KS, Mayor S (2003). Deep-orange and carnation dene
distinct stages in late endosomal biogenesis in Drosophila melanogas-
ter. J Cell Biol 161, 593–607.
Steel D, Zech M, Zhao C, Barwick KES, Burke D, Demailly D, Kumar KR,
Zorzi G, Nardocci N, Kaiyrzhanov R, etal. (2020). Loss-of-function
variants in HOPS complex genes VPS16 and VPS41 cause early onset
dystonia associated with lysosomal abnormalities. Ann Neurol 88,
867–877.
Stenmark H (2009). Rab GTPases as coordinators of vesicle trafc. Nat Rev
Mol Cell Biol 10, 513–525.
Stroupe C, Collins KM, Fratti RA, Wickner W (2006). Purication of active
HOPS complex reveals its afnities for phosphoinositides and the
SNARE Vam7p. EMBO J 25, 1579–1589.
Styers ML, Salazar G, Love R, Peden AA, Kowalczyk AP, Faundez V (2004).
The endo-lysosomal sorting machinery interacts with the intermediate
lament cytoskeleton. Mol Biol Cell 15, 5369–5382.
Swetha MG, Sriram V, Krishnan KS, Oorschot VMJ, ten Brink C, Klumperman
J, Mayor S (2011). Lysosomal membrane protein composition, acidic
pH and sterol content are regulated via a light-dependent pathway in
metazoan cells. Trafc 12, 1037–1055.
Takáts S, Glatz G, Szenci G, Boda A, Horváth GV, Hegedűs K, Kovács AL,
Juhász G (2018). Non-canonical role of the SNARE protein Ykt6 in
autophagosome-lysosome fusion. PLoS Genet 14, e1007359.
Takáts S, Pircs K, Nagy P, Varga Á, Kárpáti M, Hegedűs K, Kramer H, Kovács
AL, Sass M, Juhász G (2014). Interaction of the HOPS complex with
Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol Biol
Cell 25, 1338–1354.
Terawaki S, Vasilev F, Moriwaki T, Otomo T (2023). HOPS, CORVET and
newly-identied Hybrid tethering complexes contribute differentially
towards multiple modes of endocytosis. Sci Rep 13, 18734.
van den Boomen DJH, Sienkiewicz A, Berlin I, Jongsma MLM, van Elsland
DM, Luzio JP, Neefjes JJC, Lehner PJ (2020). A trimeric Rab7 GEF
controls NPC1-dependent lysosomal cholesterol export. Nat Commun
11, 5559.
van der Beek J, de Heus C, Liv N, Klumperman J (2022). Quantita-
tive correlative microscopy reveals the ultrastructural distribution of
endogenous endosomal proteins. J Cell Biol 221, e202106044.
van der Beek J, Jonker C, van der Welle R, Liv N, Klumperman J (2019).
CORVET, CHEVI and HOPS – multisubunit tethers of the endo-lysosomal
system in health and disease. J Cell Sci 132, jcs189134.
van der Beek J, Veenendaal T, de Heus C, van Dijk S, Ten Brink C, Liv N,
Klumperman J (Mar 31, 2023). Ultrastructural Localization of Endog-
enous LC3 by On-Section Correlative Light-Electron Microscopy. J Vis
Exp, https://doi.org/10.3791/65067.
Van Der Kant R, Jonker CTH, Wijdeven RH, Bakker J, Janssen L,
Klumperman J, Neefjes J (2015). Characterization of the mammalian
CORVET and HOPS complexes and their modular restructuring for
endosome specicity. J Biol Chem 290, 30280–30290.
van der Welle REN, Jobling R, Burns C, Sanza P, van der Beek JA,
Fasano A, Chen L, Zwartkruis FJ, Zwakenberg S, Grifn EF, etal. (2021).
24 | J. van der Beek et al. Molecular Biology of the Cell
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-
dependent TFEB/TFE3 regulation. EMBO Mol Med 13, e13258.
van Meel E, Klumperman J (2014). TGN exit of the cation-independent
mannose 6-phosphate receptor does not require acid hydrolase
binding. Cell Logist 4, e954441.
van Weert AW, Dunn KW, Geuze HJ, Maxeld FR, Stoorvogel W (1995).
Transport from late endosomes to lysosomes, but not sorting of integral
membrane proteins in endosomes, depends on the vacuolar proton
pump. J Cell Biol 130, 821–834.
Vietri M, Radulovic M, Stenmark H (2020). The many functions of ESCRTs.
Nat Rev Mol Cell Biol 21, 25–42.
Waguri S, Dewitte F, Le Borgne RL, Rouillé Y, Uchiyama Y, Dubremetz J-F,
Hoack B (2003). Visualization of TGN to endosome trafcking through
uorescently labeled MPR and AP-1 in living cells. Mol Biol Cell 14,
142–155.
Wang CW, Stromhaug PE, Kauffman EJ, Weisman LS, Klionsky DJ (2003).
Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during
the tethering/docking stage. J Cell Biol 163, 973–985.
Wang T, Yan L, Wang L, Sun J, Qu H, Ma Y, Song R, Tong X, Zhu J, Yuan Y,
etal. (2023). VPS41-mediated incomplete autophagy aggravates
cadmium-induced apoptosis in mouse hepatocytes. J Hazard Mater
459, 132243.
Wartosch L, Günesdogan U, Graham SC, Luzio JP (2015). Recruitment of
VPS33A to HOPS by VPS16 is required for lysosome fusion with endo-
somes and autophagosomes. Trafc 16, 727–742.
Wegener CS, Malerød L, Pedersen NM, Prodiga C, Bakke O, Stenmark H,
Brech A (2010). Ultrastructural characterization of giant endosomes
induced by GTPase-decient Rab5. Histochem Cell Biol 133, 41–55.
Wurmser AE, Sato TK, Emr SD (2000). New component of the vacu-
olar class C-Vps complex couples nucleotide exchange on the Ypt7
GTPase to SNARE-dependent docking and fusion. J Cell Biol 151,
551–562.
Yadati T, Houben T, Bitorina A, Shiri-Sverdlov R (2020). The ins and outs of
cathepsins: physiological function and role in disease management.
Cells 9, 1679.
Yang C, Wang X (2021). Lysosome biogenesis: regulation and functions.
J Cell Biol 220, e202102001.
Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y (1991). Balomy-
cin A1, a specic inhibitor of vacuolar-type H+-ATPase, inhibits acidica-
tion and protein degradation in lysosomes of cultured cells. J Biol Chem
266, 17707–17712.
Yu L, Chen Y, Tooze SA (2018). Autophagy pathway: Cellular and molecular
mechanisms. Autophagy 14, 207–215.
Zhang S, Tong M, Zheng D, Huang H, Li L, Ungermann C, Pan Y, Luo H,
Lei M, Tang Z, etal. (2023). C9orf72-catalyzed GTP loading of Rab39A
enables HOPS-mediated membrane tethering and fusion in mammalian
autophagy. Nat Commun 14, 6360.
Zick M, Wickner W (2013). The tethering complex HOPS catalyzes assembly
of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes.
Mol Biol Cell 24, 3746–3753.
