Receptor-mediated endocytosis is a process by which ligands that
interact with a cell surface receptor are internalized via a clathrin-
coated vesicle and undergo a series of processing steps. Soon after
internalization, receptor–ligand complexes reside in an early
endocytic vesicle that acidifies, resulting in dissociation of the
complex. Subsequently, the vesicle undergoes fission, generating a
late endocytic vesicle containing most of the ligand and a second
daughter vesicle containing most of the receptor that recycles to
the cell surface (Bananis et al., 2000; Murray et al., 2008; Pandey,
2009). The late endocytic vesicle targets the ligand to lysosomes
for degradation. The liver has an elaborate endocytic machinery
responsible for the uptake of various proteins and lipids from the
blood, which are subsequently processed and transported to the
bile (Schroeder and McNiven, 2009). We have used the hepatocyte-
specific asialoglycoprotein receptor (ASGPR) as a prototype of the
typical receptor-mediated endocytic pathway (Murray et al., 2008;
Murray and Wolkoff, 2003; Shepard et al., 2009; Stockert, 1995).
Our previous studies have shown that within 5 minutes of portal
venous injection of fluorescent ligand asialoorosomucoid (ASOR)
into rat liver, ASOR-containing vesicles are predominantly early
endocytic vesicles that mature into late endocytic vesicles by 15
minutes (Bananis et al., 2003; Bananis et al., 2004; Murray et al.,
2000). This processing of early endocytic vesicles to late endocytic
vesicles requires an intact microtubular cytoskeleton (Gruenberg et
al., 1989; Loubery et al., 2008; Soldati and Schliwa, 2006; Wolkoff
et al., 1984). In previous studies, we and others identified a number
of proteins that are required for endocytic trafficking (Jordens et
al., 2001; Murray and Wolkoff, 2005; Murray and Wolkoff, 2007;
Nath et al., 2007; Stenmark, 2009). However, it is clear that the
endocytic process is complex, and proteins that mediate and regulate
activity of this pathway have not yet been elucidated fully.
To discover new endocytic-vesicle-associated proteins, we
performed proteomic analysis of rat liver endocytic vesicles loaded
in vivo with Alexa-Fluor-488–ASOR and purified by flow
cytometry (Bananis et al., 2004). A total of 533 vesicle-associated
proteins were identified, some which were found only in early or
late vesicles, and some common to both. Several endocytic-vesicle-
associated Rab proteins were identified in this study. The Rab
proteins are small GTPases that have a regulatory role in many
aspects of vesicular transport, including vesicle budding, uncoating,
motility and fusion (Somsel Rodman and Wandinger-Ness, 2000;
Stenmark, 2009). They function by switching between GDP-bound
and GTP-bound states that regulate interactions with other proteins
known as effectors. Rab5 has been shown to be involved in the
endocytosis of several substrates such as transferrin (Bucci et al.,
1992; Nielsen et al., 1999), insulin (Fiory et al., 2004) and the
epidermal growth factor receptor (Lanzetti et al., 2000) by
regulating motility of early endosomes on microtubules (Nielsen
et al., 1999). In studies of early endocytic vesicles containing
fluorescent ASOR we found little association with Rab5 but rather
association with Rab4 (Bananis et al., 2003). Rab4 has been found
to be involved in rapid recycling of transferrin receptors and
glycosphingolipids, although the mechanistic function of this
protein is unclear (Grant and Donaldson, 2009; van der Sluijs et
al., 1992). Late endocytic vesicles contain little Rab4 or Rab5 but
instead contain Rab7 (Bananis et al., 2004; Feng et al., 1995),
which is also present on lysosomes (Zhang et al., 2009). Such Rab
Texas-Red–asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic
analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present
study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with
stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody
reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a
antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced
in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-
directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed
motility largely by recruiting Kifc1 to early endocytic vesicles.
Key words: Proteomics, Rab1a, Endocytosis
Accepted 26 October 2010
Journal of Cell Science 124, 765-775
© 2011. Published by The Company of Biologists Ltd
Proteomic analysis of endocytic vesicles: Rab1a
regulates motility of early endocytic vesicles
Aparna Mukhopadhyay1,2, Edward Nieves3, Fa-Yun Che3, Jean Wang1,2, Lianji Jin4, John W. Murray1,2,
Kristie Gordon5, Ruth Hogue Angeletti4and Allan W. Wolkoff1,2,6,*
1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
3Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
4California State University, 5241 North Maple Avenue, Fresno, CA 93710, USA
5Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA
6Division of Gastroenterology and Liver Diseases, Albert Einstein College of Medicine, Bronx, NY 10461, USA
*Author for correspondence (firstname.lastname@example.org)
Journal of Cell Science
conversion has been proposed to be a mechanism by which an
early endocytic vesicle matures to a late vesicle (Rink et al., 2005;
Rivera-Molina and Novick, 2009) and confers directionality to the
Rab1a was identified in our proteomics survey to be associated
with both early and late endocytic vesicles. The current study
aimed to characterize its potential role in endocytosis. There are
two isoforms of Rab1, Rab1a and Rab1b, which share 92% amino
acid sequence homology (Touchot et al., 1989) and are thought to
be functionally redundant in mammalian cells (Tisdale et al., 1992).
A role for Rab1 in ER-to-Golgi trafficking has been described
previously. Specifically, Rab1 recruits the tethering factor p115
into a cis-SNARE complex that programs coat protein II (COPII)
vesicles budding from the ER for fusion with the Golgi (Allan et
al., 2000) with the help of the cis-Golgi tethering protein GM130
complexed to GRASP65 (Moyer et al., 2001). Recently, a role of
Rab1a in early-endosome-to-Golgi trafficking has been reported
(Sclafani et al., 2010) and Rab1a has been described as a component
of transcytotic vesicles (Jin et al., 1996). However, a function for
this protein in endocytosis has not been described. In the present
study, using siRNA technology to knockdown Rab1a, we studied
processing of endocytic vesicles on microtubules in vitro in a
reconstituted system (Murray et al., 2000; Murray et al., 2002;
Murray and Wolkoff, 2003) and showed that Rab1a is important
for transport of early endocytic vesicles along microtubules. Using
polarity-marked microtubules, we observed that Rab1a is involved
in minus-end-directed motility of early endocytic vesicles. Our
previous studies have characterized the motors used in the
trafficking of vesicles containing ASOR. Plus-end-directed motility
of ASOR-containing early endocytic vesicles prepared from rat
and mouse liver is mediated by the conventional kinesin Kif5B
(standardized nomenclature, kinesin-1) (Lawrence et al., 2004;
Miki et al., 2005; Miki et al., 2003). However, minus-end-directed
motility is usually powered by dynein or a small group of minus-
end-directed kinesin proteins (Soldati and Schliwa, 2006). Early
endocytic vesicles prepared from rat liver use the kinesin motor
Kifc2 (Bananis et al., 2003; Bananis et al., 2004), whereas similar
vesicles prepared from mouse liver associate with and use the
kinesin motor Kifc1 (Nath et al., 2007). Late endocytic vesicles do
not use minus-end-directed kinesin motors, but instead use dynein
for motility towards the microtubule minus ends (Bananis et al.,
2004). In the present study we demonstrate that Rab1a regulates
minus-end-directed motility of early endosomes by recruiting the
minus-end kinesin Kifc1.
Purification of early and late endocytic vesicles for
Early and late endocytic vesicles containing Alexa-Fluor-488–
ASOR were prepared from rat liver and purified by flow cytometry
(Bananis et al., 2004). The presence of markers for early endosomes
(the asialoglycoprotein receptor and Rab4) (Bananis et al., 2003)
and late endosomes (Rab7 and dynein) (Bananis et al., 2004) were
confirmed by immunofluorescence (data not shown). Proteins in
each group of endocytic vesicles were resolved on one-dimensional
SDS-PAGE and subjected to analysis by nano LC ESI-MS/MS.
Identification and validation of proteins associated with
early and late endocytic vesicles
Using the criteria of one or more unique peptide hits each with
significant scores (P<0.05), 479 proteins were identified in early
endocytic vesicles and 271 proteins in late endocytic vesicles. Of
these, 217 proteins were common to both early and late vesicles
(detailed in supplementary material Tables S1–S3). We identified
a number of proteins that are integral membrane proteins or have
been implicated in trafficking of membrane proteins, including
several Rab GTPases. These selected proteins appear in Tables 1–
3. The proteins listed in these tables were identified by virtue of
correspondence of two or more significant peptides to the protein
A group of four proteins consisting of ASGPR, Rab1a, Rab14
and Rab18 that were identified in the purified vesicle preparations
were further analyzed. There was substantial colocalization of the
Rab proteins with ASOR-containing early and late endocytic
vesicles, whereas the ASGPR was largely in early endocytic vesicles
(Fig. 1). In this study, we focused on functional aspects of Rab1a
in endocytic trafficking. As seen in Fig. 1, 52% (n290) of early
and 36% (n591) of late endocytic vesicles containing ASOR were
associated with Rab1a. Conversely, only 6% (n4297) of Rab1a-
containing vesicles colocalized with ASOR.
Early endocytic vesicles associated with Rab1a do not
Mass spectrometric analysis also identified p115 as one of the
components of early endocytic vesicles. As this was not a
quantitative survey, additional studies were performed. To
validate the presence of p115 and determine its importance in
endocytosis, early endocytic vesicles were immunostained for
p115 in addition to Rab1a using specific antibodies and
colocalization was quantified. Although vesicles positive for
both p115 and Rab1a were seen [52% of the Rab1a vesicles
(n2213) contained p115, Fig. 2A], because the endosomal
preparation contained other organellar membranes, including
those from the Golgi and ER, few (6%, n194) endocytic vesicles
Interestingly, 84% (n194) of the ASOR vesicles containing
Rab1a excluded p115, indicating that in endosomes containing
Rab1a, p115 is unlikely to be an effector.
colocalized with p115.
Rab1a-associated early endocytic vesicles are also
associated with Rab4
Our previous studies showed that 80% of ASOR-containing early
endocytic vesicles are associated with Rab4 (Bananis et al., 2003).
By contrast, late endocytic vesicles contain little Rab4 but are
instead associated with Rab7 (Bananis et al., 2004). We examined
the distribution of Rab4 in Rab1a-positive ASOR-containing early
endocytic vesicles using Rab4-specific antibody in addition to anti-
Rab1a. These studies revealed that 86% (n954) of the Rab1a-
containing early endosomes were also associated with Rab4 (Fig.
2B) but only 44% of Rab4-containing early endocytic vesicles
were also associated with Rab1a.
siRNA knockdown of Rab1a
To investigate a role for Rab1a in endocytic vesicle trafficking,
studies were performed in the human hepatoma cell line Huh7 in
which Rab1a expression was knocked down using siRNA
technology. These cells express the asialoglycoprotein receptor
and have been used in a number of previous studies of endocytic
trafficking (Huang et al., 2002; Stockert et al., 2007; Treichel et
al., 1994). In case Rab1a knockdown proved toxic to the cells, a
Tet-inducible system was chosen that required the preparation of a
cell line expressing Tet repressor (TR).
766 Journal of Cell Science 124 (5)
Journal of Cell Science
Plasmids containing the siRNA target sequence of Rab1a were
transfected into the TR cell line and stable clones were selected
and picked in the presence of puromycin. These cell lines were
called 223 and 372, which refers to the nucleotide in the coding
region of Rab1a used as target sequence for knockdown. A control
cell line (PS) was also created by transfection with the empty
pSuperior vector. siRNA was induced in each of the clones in the
presence of 0.1 g/ml doxycycline and lysates were immunoblotted
for Rab1a. As a control, lysates from uninduced cells were used. It
was observed that even in the absence of doxycycline induction,
there was no detectable expression of Rab1a in the clones
containing the siRNA sequences (Fig. 3A). These results implied
Rab1a regulates endocytic trafficking
Table 2. Selected proteins identified in late but not early endocytic vesiclesa
Protein name Accession No. Mass (Da) % Coverage No. of unique peptides
Long-chain fatty acid CoA ligase 6 P33124 78,130 3.4 2
Tubulin 6 chain Q6AYZ1 49,905 8 2
Vesicle-associated membrane protein 8 Q9WUF4 11,313 24 2
aSelected proteins of interest each containing at least two significant peptides in early and late endocytic vesicles.
Table 3. Selected proteins identified in both early and late endocytic vesiclesa
% Coverage No. of unique peptides
Protein name Accession No. Mass (Da) EV LV EV LV
Asialoglycoprotein receptor 1 P02706 32,697 25.8 21.2 7 6
MRP2 Q63120 173,274 9.9 3.3 6 2
Clathrin heavy chain P11442 191,477 30.6 15.8 40 10
Golgin subfamily A member 5 Q3ZU82 82,284 22.1 7.7 10 3
Myosin-9 Q62812 226,066 41.3 35 62 51
Polymeric-immunoglobulin P15083 84,745 29.8 8.6 17 4
Ras-related protein Rab11A P62494 24,247 35.3 19.1 6 3
Ras-related protein Rab14 P61107 23,912 25.1 12.1 3 2
Ras-related protein Rab1A Q6NYB7 22,532 34.8 41.2 5 5
Ras-related protein Rab2A P05712 23521 19.3 14.2 3 2
Ras-related protein Rab6A Q9WVB1 15,763 37.9 37.9 4 2
Ras-related protein Rab7 P09527 23,489 21.7 28 3 5
Serotransferrin precursor P12346 76,314 40 11.2 20 6
Sodium/potassium-transporting P06685 112,982 20.9 8.9 18 5
ATPase 1 chain precursor
Syntaxin-7 O70257 29,643 11.5 8.1 2 2
Transferrin receptor protein 1 Q99376 70,109 17.5 11.4 10 5
aSelected proteins of interest each containing at least two significant peptides in early and late endocytic vesicles. EV, early vesicles; LV, late vesicles.
Table 1. Selected proteins identified in early but not late endocytic vesiclesa
Protein name Accession No. Mass (Da) % Coverage No. of unique peptides
Annexin A2 Q07936 38,523 7.1 3
Annexin A5 P14668 35,591 11 2
Annexin A6 P48037 75,575 9.2 4
Clathrin light chain A P08081 26,964 6 2
General vesicular transport factor p115 P41542 107,096 10.7 6
Low affinity immunoglobulin gamma Fc region receptor II precursor Q63203 32,027 23.9 5
Lysosome membrane protein IIb P27615 53,925 6.5 4
Myosin Ibb Q05096 131,835 2.5 2
PDZ domain-containing protein 1 Q9JJ40 56,765 8.8 3
Ras-related protein Rab1B P10536 22,149 31.3 3
Ras-related protein Rab18b Q5EB77 22,962 5.3 3
Ras-related protein Rab1b precursorb Q62636 20,785 6 2
Single Ig IL-1-related receptor Q4V892 46,142 22.2 5
Sodium/potassium-transporting ATPase 1 chainb P07340 35,179 8.2 3
Solute carrier organic anion transporter family member 1A1 P46720 74,129 12.2 3
Solute carrier organic anion transporter family member 1A4b O35913 73,203 3.3 7
Solute carrier organic anion transporter family member 1B2b Q9QZX8 72,719 2 3
Solute carrier organic anion transporter family member 2B1 Q9JHI3 74,166 3.5 2
Sulfate anion transporter 1 P45380 75,399 7.7 3
Vesicle-associated membrane protein-associated protein A Q9Z270 27,206 11.6 2
Vimentin P31000 53,569 35.9 12
aSelected proteins of interest each containing at least two significant peptides in early and late endocytic vesicles. Mass, molecular mass of protein (Dalton); %
Coverage, percentage coverage of the peptides of the whole protein. bProteins listed in both early and late vesicles but in late vesicles only one significant peptide
Journal of Cell Science
that the Tet-inducible system was leaky, but growth and
morphologic appearance of the Rab1a knockdown (KD) cells were
the same as those of PS and Huh7 cells. Consequently, these cells
were used for further studies in the absence of doxycycline
treatment. Expression of Rab1b and Rab4 in these cells was
unchanged (Fig. 3B), indicating specific knockdown of Rab1a.
Golgi and ER morphology is normal in Rab1a KD cells
As Rab1a has been described as a protein involved in ER-Golgi
trafficking, we examined whether its knockdown would affect
the appearance of these organelles. As seen in Fig. 3C,
morphology of the Golgi (p115, in green) and ER (PDI, in red)
in the two Rab1a KD cell lines did not differ from the empty
vector (PS) cell line.
ASOR trafficking is perturbed in Rab1a KD cells
To test the role of Rab1a in endocytic trafficking in cultured cells,
live-cell imaging following fluorescent ASOR uptake in Rab1a
KD cells was performed. Fig. 4A is a composite panel of
representative images, showing time-dependent accumulation of
fluorescence as vesicles containing endocytosed fluorescent ligand
grow larger and brighter in the Rab1a KD cells in contrast to the
control PS cells. Images from three independent experiments were
quantified where the maximum and minimum pixel intensity values
of all the images were set and the mean pixel intensity of individual
cells measured by ImageJ. To normalize values between
experiments and different cell lines, the pixel intensity at time 0
was set at 1 and the values of the other time points calculated
accordingly. These data, represented graphically in Fig. 4B, show
the increase in pixel intensity of fluorescence in the Rab1a KD
cells, indicating accumulation of protein. By contrast, the PS cells
did not get brighter over time, indicating that endocytosed ASOR
is efficiently processed and degraded in these cells.
To determine directly whether there is a defect in intracellular
ASOR processing in Rab1a KD cells, surface binding and
degradation of [125I]ASOR was assayed. In these studies,
[125I]ASOR was bound to the surface of cells at 4°C, excess was
washed away and the cells were shifted to 37°C to initiate
endocytosis. Normally, endocytosed ASOR is delivered to
lysosomes for degradation. Degraded protein is quantified through
measurement of acid soluble radioactivity in the medium, whereas
surface bound ligand is estimated from radioactivity released with
20 mM EGTA. In the control PS cells, we observed that 34% of
[125I]ASOR that had been bound to the cell surface at time 0 was
degraded by 90 minutes (Fig. 4C). Degradation was significantly
reduced in the Rab1a KD cell line 223 to 18% of that initially
bound to the surface (P<0.02), consistent with impaired delivery
of ligand to lysosomes in the absence of Rab1a. Because all
experiments with the two Rab1a KD cell lines yielded similar
results, data from further experiments using the two cell lines were
combined and are represented as Rab1a KD. Similarly, results of
Huh7 and PS were combined as a control because both of these
cell lines yielded identical results.
Rab1a antibody reduces microtubule-based motility of
early but not late endocytic vesicles
Previously, we showed that early and late endocytic vesicles can
attach to and move along microtubules in vitro (Bananis et al., 2000;
Bananis et al., 2003; Bananis et al., 2004; Murray et al., 2000;
Murray et al., 2002; Murray et al., 2008; Murray and Wolkoff, 2005;
Murray and Wolkoff, 2007) and this trafficking is an essential part
of the endocytic processing of ASOR (Novikoff et al., 1996). Slower
processing (Fig. 4B) and reduced degradation (Fig. 4C) of ASOR in
the absence of Rab1a could be due to reduced motility of vesicles as
they traffic via microtubules to reach lysosomes. Initial experiments
examined the effect of Rab1a antibody preincubation on motility of
768 Journal of Cell Science 124 (5)
Fig. 1. Validation of proteins identified by proteomic analysis of FACS purified early and late endocytic vesicles. Texas-Red–ASOR-containing early (left
panel) and late (right panel) endocytic vesicles were stained for selected proteins as indicated using specific antibodies. The images were pseudocolored and
merged using ImageJ. Colocalization was quantified and is indicated as a percentage of vesicles containing ASOR. Scale bars: 10m.
Journal of Cell Science
endocytic vesicles on microtubules. In these studies, motility of
endocytic vesicles containing Texas-Red–ASOR on microtubules
was assayed following preincubation of vesicles with anti-Rab1a
antibody. Control studies were performed in the absence of antibody
and in the presence of nonimmune rabbit IgG. In the absence of
antibody, 44% (n318) of early endocytic vesicles on microtubules
were motile (Fig. 5A). By contrast, only 25% (n303, P<0.001) of
early endocytic vesicles moved on microtubules following
preincubation with Rab1a antibody. This was not due to a non-
specific effect of IgG, because motility of these vesicles was enhanced
in the presence of non-immune IgG (Fig. 5A). Interestingly, there
was no effect on the motility of late endocytic vesicles upon
preincubation with Rab1a antibody (Fig. 5A).
Early endocytic vesicles prepared from Rab1a KD cells
have reduced microtubule-based motility
To further establish a role for Rab1a in microtubule-based motility,
we prepared early endocytic vesicles containing Alexa-Fluor-488–
ASOR from the Rab1a KD and control cells and used them in
motility assays similar to that described above. Of these vesicles
prepared from control and KD cells, 70% contained the ASGPR
receptor as assessed by immunostaining, indicating that they are
largely early endocytic vesicles (data not shown). As seen in Fig. 5B,
in the absence of Rab1a, fewer (30–35%, P<0.001) vesicles moved
on microtubules compared with those prepared from the control PS
cells (51%). A representative movie of motility of vesicles prepared
from Huh7 cells is shown in supplementary material Movie 1.
Rab1a is required for minus-end-directed movement of
early endocytic vesicles on microtubules
The studies presented above show that Rab1a is required for
optimal motility of early endocytic vesicles on microtubules. In
Rab1a regulates endocytic trafficking
Fig. 2. Texas-Red–ASOR-containing early endocytic vesicles that
colocalize with Rab1a contain Rab4 but not p115. Texas-Red–ASOR-
containing early endocytic vesicles were stained for Rab1a and either p115 (A)
or Rab4 (B) using specific antibodies. Images were pseudocolored and merged
using Image J. Arrows in A indicate vesicles containing Rab1a and Texas Red,
but excluding p115. Arrows in B indicate vesicles containing Texas Red,
Rab1a and Rab4. Scale bars: 10m.
Fig. 3. SiRNA knockdown of Rab1a. Huh7 cells were stably transfected
with siRNA designed to knockdown Rab1a. (A,B)Lysates from parental
Huh7, empty pSuperior transfected (PS) cell lines and those from different
clones from the two Rab1a KD cells lines, 223 and 372 (Rab1a KD) were
subjected to immunoblotting for Rab1a (A), Rab1b or Rab4 (B) as indicated.
The gels were also probed for actin as a loading control. Position of molecular
size markers (in kDa) is indicated. (C)ER and Golgi morphology in Rab1a
KD cells. Rab1a KD cell lines, 223 and 372 as well as control (PS) cells were
grown on MatTek plates and stained for the Golgi marker p115 (green) and the
ER marker PDI (red). Single channel images were merged and pseudocolored
using ImageJ software. Panels on the right represent the bright-field images of
panels to the left. Scale bars: 50m.
Journal of Cell Science
further studies, we examined whether all movement was reduced
or whether there was a specific reduction in movement towards the
plus or minus ends of microtubules. To examine this question,
motility assays were performed with polarity-marked microtubules
and the number of vesicles moving towards the plus and the minus
ends were quantified. An example of vesicle motility on polarity
marked microtubules is shown in supplementary material Movie 2.
Similarly to previous results (Bananis et al., 2000), it was observed
that almost equal numbers of early endocytic vesicles moved
towards the plus and minus ends (27% and 30% of total vesicles,
respectively, Fig. 6A). Upon preincubation with Rab1a antibody,
plus-end-directed motility remained the same (32%), but minus-
end-directed motility diminished significantly (11% of total
vesicles, P<0.001). To further establish a requirement for Rab1a in
minus-end-directed motility, we used early endocytic vesicles from
Rab1a KD cells and quantified their directional motility. Similarly
to results in the presence of Rab1a antibody, the vesicles from
Rab1a KD cells had reduced motility (12% of total vesicles,
P<0.001) towards the minus end of microtubules when compared
with the control (28% of total vesicles, Fig. 6B).
Rab1a regulates minus-end-directed motility of early
endocytic vesicles by recruiting Kifc1
A defect in minus-end-directed motility of vesicles lacking Rab1a
indicated that activity of a minus-end-directed motor could be
regulated by Rab1a. Our previous work has characterized the plus
and minus end motors driving motility of endocytic vesicles derived
from rat and mouse livers (Bananis et al., 2000; Bananis et al.,
2003; Bananis et al., 2004; Murray et al., 2000; Murray and
Wolkoff, 2003; Nath et al., 2007). As the Rab1a KD cells are
derived from the human hepatoma cell line Huh7, we examined
which minus-end-directed motor is used by these vesicles, because
they were previously uncharacterized. Early endocytic vesicles
containing Alexa-Fluor-488–ASOR were prepared from the Rab1a
KD and control cell lines and immunostained for dynein, Kifc1
770 Journal of Cell Science 124 (5)
Fig. 4. Rab1a KD cells exhibit impaired processing and degradation of
ASOR. The Rab1a KD cell lines (223 and 372) as well as the vector-
transfected control cells (PS) were grown on MatTek plates and were used for
a study where uptake of Alexa-Fluor-488–ASOR prebound to the cell surface
was followed for 80 minutes (min) by live-cell imaging. (A)Representative
images. For clarity in print, the brightness and contrast of these images were
enhanced beyond the adjustments described in B. Scale bar: 5m.
(B)Graphical representation of average pixel intensity of Alexa-Fluor-488–
ASOR in cells over 80 minutes. Pixel intensities of all images from three
independent experiments were set to the same maximum and minimum level
and the mean gray value of individual cells were measured using ImageJ. To
normalize between different cell lines and experiments, the value at 0 minutes
was set at 1.0 and all other values calculated accordingly by dividing the mean
intensity at each time point with the mean intensity at time 0 minutes. Error
bars represent s.d. from the mean. (C)Degradation of [125I]ASOR in Rab1a
KD (223) and control (PS) cells. [125I]ASOR was initially bound to cells on ice
and then allowed to be taken up by endocytosis by shifting the cells to 37°C.
Degraded ASOR was measured by counting acid soluble radioactivity in the
medium at the end of 90 minutes. In replicate plates, surface-bound ASOR at
0 minutes was released in 20 mM EGTA and counted. Degradation at 90
minutes was calculated as a percentage of the total amount of ASOR that was
surface bound at 0 minutes. Error bars represent s.d. *P<0.02.
Fig. 5. Rab1a regulates motility of early endocytic vesicles on
microtubules. (A)Texas-Red–ASOR-containing early (EE) and late (LE)
endocytic vesicles were attached to Rhodamine-labeled microtubules and their
motility in the presence (+Ab) or absence (–Ab) of Rab1a antibody was
studied. A control experiment was performed in the presence of normal rabbit
IgG (IgG). (B)Motility of early endocytic vesicles prepared from Rab1a KD
(223 and 372) and control cells (PS and Huh7). The percentage of
microtubule-bound vesicles that moved upon addition of 50M ATP is shown.
In both panels, the number of total vesicles counted is in parentheses.
Journal of Cell Science
and Kifc2 using specific antibodies (Fig. 7A). Represented
graphically in Fig. 7B is a quantification of the amount of
colocalization observed between the motor proteins with Alexa-
Fluor-488–ASOR. Among the vesicles prepared from the control
cells, 47% colocalized with dynein, 36% with Kifc1 and 23% with
Kifc2. Similar numbers of vesicles prepared from the Rab1a KD
cells colocalized with dynein (45%) and Kifc2 (23%). Interestingly
however, these vesicles exhibited an almost complete lack of
colocalization (6%, P<0.001) with Kifc1. These data indicate that
Rab1a is essential for the recruitment of Kifc1 to early endocytic
In the present study, fluorescent ASOR-containing early and late
endocytic vesicles were highly enriched using fluorescence sorting
and subjected to proteomic analysis. A number of transporters and
potential regulatory molecules were identified. The presence of the
organic anion transporters, transferrin receptor, polymeric
immunoglobulin receptor, asialoglycoprotein receptor and Na+/K+-
transporting ATPase in the early endocytic vesicles indicate that
many proteins that are endocytosed from the cell surface use the
same vesicle, at least initially. It has been observed previously that
45% of ASOR vesicles colocalize with the bile acid transporter,
Na+-taurocholate co-transporting polypeptide (NTCP) and 65% of
the NTCP vesicles colocalize with transferrin receptor (Sarkar et
al., 2006), indicating that a population of NTCP and transferrin
could possibly use the same pathway as ASOR for uptake from the
cell surface. ASGPR1 and clathrin (Murray and Wolkoff, 2003;
Stockert, 1995; Wolkoff et al., 1984), which are known to be
present exclusively in early endosomes, were also identified in late
endocytic vesicles. This could be due to partial contamination of
late endocytic vesicles with early vesicles, because preparation of
vesicles involves the injection of fluorescent ASOR into the portal
vein of rats, which, despite high first-pass extraction, still exhibits
a component of non-synchronous uptake into the liver.
Rab1a regulates endocytic trafficking
Fig. 6. Rab1a regulates minus-end-directed motility of early endocytic
vesicles. Polarity marked microtubules were prepared and motility of (A)
Texas-Red–ASOR-containing early endocytic vesicles in the presence (+Ab)
and absence (–Ab) of Rab1a antibody and (B) Alexa-Fluor-488–ASOR-
containing vesicles prepared from the Rab1a KD (223 and 372) and control
cells (PS and Huh7) were studied. The percentage of total vesicles moving
towards the plus and minus ends was scored and is represented graphically.
The numbers in parentheses indicate the total number of vesicles attached to
polarity marked microtubules that were counted. *P<0.001.
Fig. 7. Rab1a recruits the minus-end-directed kinesin motor Kifc1 to early
endocytic vesicles. Alexa-Fluor-488–ASOR-containing early endocytic
vesicle were prepared from Rab1a KD (223 and 372) and control PS cells and
stained with antibody against dynein, Kifc1 or Kifc2. (A)A panel of
representative images showing colocalization of early endocytic vesicles from
the cell lines with the indicated motor proteins. Arrowheads indicate ASOR-
containing vesicles colocalizing with the indicated motor protein. Scale bars:
10m. (B)Graphical representation of the percentage colocalization. Rab1a
KD, black bars; PS, gray bars. The numbers in parentheses indicate the total
number of vesicles examined. *P<0.001.
Journal of Cell Science
Several potential regulatory molecules such as annexin-2,
annexin-5 and annexin-6, syntaxin-7 and Rab1a, Rab1b, Rab2A,
Rab6A, Rab7, Rab8B, Rab11A, Rab14 and Rab18 were also
detected. The Rab proteins that have been described to have a role
in various stages of endocytic trafficking are Rab4, Rab5, Rab7,
Rab9, Rab15 and Rab22 (Stenmark, 2009), and this study reveals
a previously unknown function of Rab1a in this process. Previously
we reported that 80–90% of Texas-Red–ASOR-containing early
endocytic vesicles prepared from rat liver contain Rab4 (Bananis
et al., 2003). In the present study, we show that a similar percentage
of ASOR vesicles associated with Rab1a also contained Rab4.
However, only 44% of ASOR vesicles containing Rab4 were
associated with Rab1a. This could indicate that Rab4 associates
with early endocytic vesicles before Rab1a and might be involved
in the recruitment of Rab1a to these vesicles. There are several
reports that describe possible mechanisms of how multiple Rab
proteins function and coordinate with each other on the same
vesicle (Del Conte-Zerial et al., 2008; Fukuda et al., 2008; Markgraf
et al., 2007; Rivera-Molina and Novick, 2009; Sonnichsen et al.,
2000; Spang, 2009).
Rab1 has been implicated in regulation of COPII vesicle fusion
to the cis-Golgi in association with p115, GM130 and GRASP65
(Allan et al., 2000; Moyer et al., 2001). Rab1a has also been
described as a component in the transcytotic pathway in rat liver
(Jin et al., 1996). This pathway traffics cargo such as polymeric
IgA (pIgA) and its receptors from the basolateral hepatocyte
membrane to the apical side where vesicle content is released into
the bile canaliculus. These pIgA-rich transcytotic vesicles are also
associated with p115 (Jin et al., 1996; Sztul et al., 1991) and both
the polymeric immunoglobulin receptor and p115 were discovered
to be components of early endocytic vesicles. In the present study,
we report the presence of vesicles that contain endocytosed ASOR
that colocalize with Rab1a, but exclude p115 (Fig. 2A). These data
show that Rab1a can associate with specific vesicles in the absence
of p115, indicating that Rab1a uses different effectors to function
simultaneously in regulating endocytosis as well as ER-to-Golgi
Knockdown Huh7 cell lines were created for examination of the
role of Rab1a in endocytosis. These cell lines expressed Rab1b and
Rab4 and exhibited normal morphology of the Golgi and ER.
There are mixed reports on the effect of Rab1a knockdown on the
Golgi. Transient transfections into HeLa and HEK293 cells resulted
in complete fragmentation of the Golgi in some reports (Hyvola et
al., 2006; Razi et al., 2009; Winslow et al., 2010), whereas less or
no fragmentation in others (Dejgaard et al., 2008; Dumaresq-
Doiron et al., 2010). Our studies differ from these reports because
we created stable cell lines with knockdown of Rab1a, in contrast
to the use of transfected siRNAs. The simplest explanation for lack
of any fragmentation in our cells is compensation of the loss of
Rab1a by Rab1b because both isoforms have redundant functions
although there are several ways a cell could adapt itself, which
would be manifested only in a stable cell line. Dysfunction of the
secretory pathway cannot be ruled out, although it would probably
be minor considering the normal appearance and growth of these
The knockdown cell lines exhibited slower processing and
degradation of the endocytic ligand ASOR, implying that Rab1a is
involved in endocytic processing of ASOR. Because vesicle
maturation and endocytic processing require trafficking of vesicles
on microtubules, in vitro analysis of vesicle movement on
microtubules was examined. The use of an inhibitory antibody
against Rab1a in vesicle-motility assays revealed reduced motility
of early but not late endocytic vesicles. This suggests that Rab1a
has an important role in the processing of early endocytic vesicles.
Although a significant number (36%) of late endocytic vesicles
also contain Rab1a, it does not appear to be involved in motility
of these vesicles on microtubules. Studies using vesicles prepared
from Rab1a KD cells confirmed that Rab1a influences the
trafficking of early endocytic vesicles on microtubules. Hence the
slower processing and reduced degradation of ASOR observed in
these cells could be attributed to reduced motility of vesicles that
allowed ASOR to accumulate in the cells.
Experiments using polarity-marked microtubules showed that
anti-Rab1a antibody specifically inhibits minus-end-directed
motility of vesicles on microtubules. A role for Rab1a in minus-
end-directed motility was confirmed using vesicles lacking Rab1a
that were prepared from Huh7 cells lines with a stable knockdown
of Rab1a. This effect of Rab1a is topologically significant, because
movement of vesicles from the cell surface to lysosomes requires
trafficking towards the minus end of microtubules.
Minus-end-directed motility is potentially driven by two motors:
cytoplasmic dynein and C-terminal minus-end-directed kinesins,
which are members of the kinesin-14 family (Soldati and Schliwa,
2006). Among the minus-end-directed kinesins, only Kifc1 (kinesin-
14A), Kifc2 (kinesin-14B) and Kifc3 have been reported to be
involved in intracellular trafficking (Nath et al., 2007; Xu et al.,
2002; Yang and Sperry, 2003; Yang and Goldstein, 1998; Yang et
al., 2001). Our previous studies showed that ASOR-containing
early endocytic vesicles prepared from rat liver use Kifc2 (Bananis
et al., 2000; Bananis et al., 2003; Bananis et al., 2004; Murray et
al., 2000), whereas those prepared from mouse livers use Kifc1
(Nath et al., 2007) for minus-end-directed motility. Although a
substantial number of ASOR-containing vesicles colocalize with
NTCP, the NTCP vesicles use dynein for minus-end-directed
motility (Sarkar et al., 2006). In the present study,
immunofluorescence experiments using ASOR-containing early
endocytic vesicles prepared from parental Huh7 cells showed 47%
colocalization with dynein, 35% with Kifc1 and 23% with Kifc2.
The presence of dynein in these vesicles is in contrast to our earlier
observations using early endocytic vesicles from rat and mouse
liver. This could represent differences in timing of endocytic
processing between cell lines and intact liver. The minimal
colocalization of Kifc1 with ASOR-containing vesicles prepared
from Rab1a KD cells indicates that Rab1a is required for the
recruitment of the motor Kifc1 to these endocytic vesicles. This
provides an explanation for the loss of minus-end-directed motility
in vesicles lacking Rab1a.
This study indicates that although Rab1a is present in both early
and late endocytic vesicles, it is required for motility of only early
endocytic vesicles towards the minus end of microtubules by the
recruitment of Kifc1. We hypothesize that Rab1a functions as a
scaffold to which stage-specific proteins and motors can bind. This
represents a novel function of Rab1a, which is distinct from its
well-characterized function in the secretory pathway.
Materials and Methods
Cells, reagents and antibodies
The human hepatoma cell line Huh7 was maintained in RPMI medium containing
L-glutamine (Mediatech, Manassas, VA) and supplemented with 10% fetal bovine
serum (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin-streptomycin
(Mediatech). For the tetracycline-inducible system, Tet System approved serum
was purchased from Clontech Laboratories, Mountain View, CA. Tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Pierce, Rockford,
IL. Trifluoroacetic acid (TFA, protein sequencer grade) was from Applied Biosystems,
772 Journal of Cell Science 124 (5)
Journal of Cell Science
Foster City, CA. All other reagents were from Sigma-Aldrich, St Louis, MO, unless
otherwise noted. All chemicals were of analytical grade or higher.
The following antibodies were used: rabbit polyclonal antibodies against Rab1a,
Rab1b, Dynein and normal rabbit IgG were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA); mouse anti-Rab4 (BD Transduction Laboratories, San Jose, CA);
rabbit polyclonal anti-Rab14 (Abcam, Cambridge, MA); rabbit polyclonal anti-
Rab18 (Calbiochem, San Diego, CA); mouse p115 (a gift from Dennis Shields,
Albert Einstein College of Medicine, NY); rabbit anti-PDI (Stressgen, Ann Arbor,
MI); mouse anti--actin (Sigma); mouse anti-human Kifc1 (AbD Serotec, Oxford,
UK); rabbit polyclonal anti Kifc2 (Nath et al., 2007); Cy5-conjugated Goat Anti-
Rabbit IgG and Cy3-conjugated goat anti-mouse IgG (H+L) (Jackson
ImmunoResearch Laboratories, West Grove, PA).
Preparation of labeled ASOR
Asialoorosomucoid (ASOR) was prepared by hydrolysis of human 1-orosomucoid
(Sigma) in 0.1 N sulfuric acid at 75°C for 1 hour. The solution was neutralized with
1 N NaOH, dialyzed against water, and protein concentration determined as described
previously (Stockert et al., 1995). Fluorescent ASOR was prepared by conjugation
with either Texas Red sulfonyl chloride or Alexa Fluor 488 carboxylic acid,
succinimidyl ester (Molecular Probes, Eugene, OR) following the manufacturer’s
instructions. [125I]ASOR was prepared by incubating 100 g ASOR with 5 mCi
Na125I (Perkin Elmer, Boston, MA) in the presence of Pierce iodination beads
(Pierce Biotechnology, Rockford, IL) for 15 minutes. The solution was applied to a
20 ml G25 column and radioactivity in 0.5 ml fractions was determined in a gamma
counter. The early fractions containing maximum radioactivity were pooled and used
for uptake studies. A yield of 400–600 c.p.m./ng ASOR was usually achieved.
Preparation of early and late endocytic vesicles
Fluorescent ASOR containing early and late endocytic vesicles were prepared from
livers of 200–250 g male Sprague–Dawley rats (Taconic Farms, Germantown, NY)
as described previously (Bananis et al., 2003). Briefly, livers were removed either 5
minutes (for early endocytic vesicles) or 15 minutes (for late endocytic vesicles)
after injection of 50 g Texas-Red–ASOR into the portal vein. After Dounce
homogenization, a postnuclear supernatant was obtained which was then subjected
to Sephacryl S200 (Pharmacia, Uppsala, Sweden) column chromatography. Vesicle
enriched fractions (cloudy fractions) were pooled and adjusted to 1.4 M sucrose.
This was then layered at the bottom of a 1.4–1.2–0.25 M discontinuous sucrose
density gradient and subjected to centrifugation at 100,000 g for 2 hours. The
vesicles were harvested from the 1.2–0.25 M sucrose interface and stored in small
aliquots at –80°C until used. All animal procedures were approved by the Animal
Institute Committee of the Albert Einstein College of Medicine.
Early endocytic vesicles were also prepared from Huh7 cells. The cells were
incubated on ice with 1.5 g/ml Alexa-Fluor-488–ASOR in buffer (135 mM NaCl,
0.81 mM MgSO4, 1.2 mM MgCl2, 27.8 mM glucose, 2.5 mM CaCl2, 25 mM
HEPES, pH 7.2) for 1 hour. Unbound ASOR was removed by washing and the cells
were incubated at 37°C for 7 minutes to initiate endocytosis. The cells were then
washed in cold buffer, lysed by Dounce homogenization and vesicles were prepared
similarly to those from rat livers, the only difference being omission of the Sephacryl
S200 column chromatography step.
Flow cytometric purification of fluorescent early and late endocytic
Alexa-Fluor-488–ASOR-containing endocytic vesicles were subjected to sorting on
a DakoCytomation Modular Flow (MoFlo) High-Performance Cell Sorter
(DakoCytomation, Fort Collins, CO) equipped with a 488 nm coherent argon laser
and a collection/emission 530/540 nm filter. For control determinations, unlabeled
vesicles were isolated from rat livers that had not been injected with ASOR. Data
acquisition and analysis was performed using DakoCytomation MoFlo Summit
Software (DakoCytomation, Fort Collins, CO). Details and validation of this
procedure have been published (Bananis et al., 2004).
Proteomic analysis of vesicle-associated proteins
Following fluorescence sorting, approximately 3?108early or late endocytic vesicles
(70–90 g protein) were subjected to SDS-PAGE separation using 10%
polyacrylamide precast minigels (Bio-Rad, Hercules, CA). Approximately 10–15 g
of vesicle protein in sample buffer was applied to each of six lanes on separate gels
for early and late vesicles. Protein was visualized following staining for 10–15
minutes with 0.2% Coomassie Blue. Each lane was then cut into approximately 50
sequential slices of ~1 mm thickness. Corresponding slices from each of the lanes
were combined, cut into 1?1 mm pieces, and washed with water. The gel pieces
were destained in 0.2 M NH4HCO3, pH 8.9 and acetonitrile (1:1 v/v) and reduced
in 0.1 M NH4HCO3, pH 8.9 containing 10 mM DTT at 56°C. The reduced cysteine
residues were subsequently alkylated in 55 mM iodoacetamide in 100 mM NH4HCO3
and the gel pieces were then washed in acetonitrile and dried. The dried gel pieces
were treated with approximately 200 ng trypsin (sequencing grade; Promega,
Madison, WI) in 50 mM NH4HCO3, pH 8.9 on ice for 45 minutes, the excess
removed and the digestion was allowed to complete at 37°C for 18 hours in 50 mM
NH4HCO3, pH 8.9. Trifluoroacetic acid (TFA) was then added to a final concentration
of 0.1% and used for nano LC ESI-MS/MS analysis.
Nanoelectrospray LC-MS/MS analysis and protein identification
Tryptic digests were loaded and separated using the UltiMate FAMOS Switchos
nano-HPLC system (LC Packings, Dionex; Sunnyvale, CA) connected online to a
LTQ Linear Ion Trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA)
equipped with a nanospray source. The mobile phases consisted of 5% acetonitrile
in water, 0.1% formic acid (A) and 80% acetonitrile in water, 0.1% formic acid (B).
After injection (15 l sample) and loading onto a C18trap column, 0.3 mm ID ?5
mm, the tryptic peptides were separated on a C18analytical HPLC column (75 m
ID ?15 cm; Pepmap, 3 m, 100 A; LC Packings, Dionex, Sunnyvale, CA). The flow
rate for loading and desalting was 15 l/minute for 30 minutes and the analytical
separation was performed at 250 nl/minute. The gradient used was: 2% to 55% B
over 65 minutes; hold at 55% B for 10 minutes; increase to 95% B for 5 minutes
and hold at 95% B for another 5 minutes. The HPLC eluent was electrosprayed into
the LTQ using the nanospray source. After an initial MS survey scan, m/z 300–1800,
MS/MS scans were obtained from the three most intense ions using a normalized
collision energy of 35%. DTA files were generated from the raw data files, merged
and searched against the SwissProt Rattus database with Mascot (version 2.1.04).
The search parameters used were: variable modifications – N/Q deamidation, oxidized
Met, carbamidomethyl C; 1 missed cleavage; peptide mass tolerance of ±2 Da and
±0.8 Da for product ions.
Immunoblots were performed as we have described previously (Bananis et al.,
2004). In brief, presorted endocytic vesicles were subjected to SDS-PAGE (~15 g
total protein/lane) under reducing conditions (100 mM DTT) and transferred to a
PVDF membrane (Perkin Elmer, Boston, MA). The membrane was blocked with
TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.6) containing 0.1% Tween 20 and 10%
nonfat dried milk before incubation with primary antibody diluted appropriately in
TBS, 0.1% Tween 20 and 2% nonfat dried milk. Immunoblot analysis was then
performed using appropriate primary and HRP-conjugated secondary antibodies.
Immunofluorescence and motility assay of vesicles
Immunofluorescence and microtubule-based vesicle motility studies were performed
in an optical chamber as described previously (Murray et al., 2002; Murray and
Wolkoff, 2007). Vesicles were stained for selected proteins using specific antibodies
in buffer containing 35 mM PIPES-K2, 5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA,
2 mg/ml BSA, 4 mM DTT, 2 mg/ml vitamin C with 5 mg/ml casein, pH 7.4.
Fluorescent microtubules were polymerized from a 7:1 mixture of tubulin (10
mg/ml) and Rhodamine–tubulin (1.7 mg/ml) (Cytoskeleton Inc., Denver, CO) in
buffer containing 8 mM PIPES-K2, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP with
3% glycerol, pH 7.0 at 37°C. The polymerized microtubules were stabilized and
stored in this buffer supplemented with 20 M Taxol. In experiments involving
polarity marked microtubules, end labeling was achieved by initially polymerizing
‘dim’ seeds containing unlabeled and Rhodamine–tubulin at a ratio of 72:1 for 5
minutes. These seeds were sheared and ‘bright’ tubulin that had unlabeled and
rhodamine tubulin at a concentration of 6:1 in the buffer described above was added
and allowed to polymerize for 6 minutes. The polarity marked microtubules were
stored in 20 M Taxol-containing buffer and used the same day.
For motility experiments, microtubules were diluted in MT buffer (35 mM PIPES-
K2, 5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 2 mg/ml BSA, pH 7.4 with 20 M
Taxol) and flowed into an optical chamber that had been precoated with 30 g/ml
DEAE-dextran. After washing off excess microtubules, vesicles were flowed in and
allowed to bind to the microtubules at room temperature for 10 minutes in a
humidified chamber. In experiments involving addition of Rab1a antibody, vesicles
were washed in blocking buffer (35 mM PIPES-K2, 5 mM MgCl2, 1 mM EGTA, 0.5
mM EDTA, 2 mg/ml BSA, 4 mM DTT, 2 mg/ml vitamin C with 5 mg/ml casein,
pH 7.4) before addition of antibody diluted 1:50 in the same buffer. Upon completion
of antibody incubation, the chamber was washed in assay buffer (35 mM PIPES-K2,
5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 2 mg/ml BSA, 4 mM DTT, 2 mg/ml
vitamin C, pH 7.4). Images were acquired upon addition of 50 M ATP to the optical
chamber on the microscope stage heated to 37°C.
siRNA knockdown of Rab1a
A stable Huh7 cell line expressing the Tet Repressor was established by transfection
of pcDNA6/TR (Invitrogen, Carlsbad, CA) and selecting stable clones in 1.5 g/ml
blasticidine S HCl (Invitrogen, Carlsbad, CA). Individual clones were selected and
tested for expression of Tet repressor by immunoblotting using Tet repressor antibody
(MoBitec, Goettingen). The clone expressing the highest level of Tet Repressor was
Rab1a was knocked down by siRNA using a vector-based approach. Two target
sequences were selected 5?-CAATCACCTCCAGTTATTA-3? and 5?-CAAAT -
GTGATCTGACCACA-3? corresponding to nucleotides 223 and 372 in the human
RAB1A sequence (Accession No. NM_004161). Oligonucleotides containing the
sense-loop-antisense sequence were synthesized and cloned into pSuperior.Puro
(Oligoengine, Seattle, WA) following the manufacturer’s instructions. The vectors
were transfected into Huh7 cells expressing Tet repressor (TR cells). Stable clones
were selected in 2.0 g/ml puromycin (Clontech, Mountain View, CA). Individual
clones were selected and tested for Rab1a expression by immunoblotting in the
absence or presence of 0.1 g/ml doxycycline hyclate (Sigma). Clones exhibiting
Rab1a regulates endocytic trafficking
Journal of Cell Science
maximum knockdown of Rab1a expression were selected and used for subsequent
For fluorescent ASOR-uptake studies, cells were grown to confluence either on 50
mm MatTek plates (MatTek Corporation, Ashland, MA) or eight-well Labtek
chambers (Thermo Fisher Scientific, Rochester, NY). The plates were chilled on ice
and 10 g/ml Alexa-Fluor-488–ASOR in binding buffer (135 mM NaCl, 0.81 mM
MgSO4, 1.2 mM MgCl2, 27.8 mM glucose, 2.5 mM CaCl2, 25 mM HEPES, pH 7.2)
was added and allowed to bind for 1 hour on ice. Excess unbound ASOR was
removed by washing and the plates were warmed to 37°C on the microscope stage,
and images were captured over 90 minutes.
[125I]ASOR-uptake assays were performed as described previously (Stockert et
al., 2007). Briefly, cells were grown to confluence and chilled on ice before addition
of 1.2 g [125I]ASOR per plate in binding buffer. In control plates, 100 g of
unlabeled ASOR was added to estimate non-specific binding. ASOR was allowed to
bind for 1 hour on ice and the excess washed off before the plates were shifted to
37°C to initiate endocytosis. Degradation was estimated by counting acid (10%
trichloroacetic acid, 2% phosphotungstic acid) soluble radioactivity in the medium,
whereas surface-bound ASOR was released in 20 mM EGTA and counted.
Image acquisition and processing
Images were acquired with a 60? 1.4 numerical aperture Olympus objective on an
Olympus 1?71 inverted microscope containing automated excitation and emission
filter wheels maintained at 37°C. Data were collected through a CoolSNAP HQ
cooled charge-coupled device (Photometrics, Roper Scientific, Tucson, AZ) camera
regulated by MetaMorph (Molecular Devices, Sunnyvale, CA) software. Fluorescent
images were analyzed using ImageJ 1.39u (National Institutes of Health public
domain; rsb.info.nih.gov/ij/) and Adobe Photoshop CS2 version 9.0.2 (Adobe
Systems, San Jose, CA).
Colocalization of fluorescent vesicles was automatically quantified using the
Autoscore_Co-localization macro written by John W. Murray to run in ImageJ 1.39u
software. This macro is based on previous methods (Murray et al., 2002) and
functions by segmenting images of vesicles into discrete spots and quantifying the
presence of fluorescence at each spot in images of alternate fluorescence channels
of the same field. It uses the SpotEnhancing filter written by Daniel Sage (Biomedical
Imaging Group, Lausanne, Switzerland) and the Analyze Particles function of ImageJ
1.39u. Threshold intensity is chosen automatically based on image intensity and
For microtubule-based motility studies, time-lapse movies were taken at 1 frame
per second for 90 seconds. Movies were analyzed using ImageJ 1.39u software by
scoring the number of vesicles on microtubules and manually counting those that
exhibited movement. Vesicles were considered motile if they were observed to move
from their original position along a microtubule over a span of two or more frames.
Vesicles that were not on microtubules or those that flowed away upon addition of
ATP were not scored. In experiments involving use of polarity marked microtubules,
only those vesicles on microtubules where the polarity could be distinguished were
Live cell fluorescent ASOR uptake images at different time points were first
normalized by setting the maximum and minimum pixel intensity values within the
same limits using ImageJ 1.39u. The mean gray value of individual cells was
measured and an average calculated for each time point. In order to normalize the
intensity of fluorescence between different cell lines, the intensity was set at 1.0 for
time 0 minutes and that of all the other time points were calculated accordingly by
dividing the mean intensity value at each time point by the intensity value at time 0
Statistical analysis was performed using Chi-square or Student’s t-test as appropriate
using Microsoft Excel 2000.
This work was supported by National Institutes of Health grants
DK41918, DK07218 and DK041296. Deposited in PMC for release
after 12 months.
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Rab1a regulates endocytic trafficking
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