Diverse functions of myosin VI in spermiogenesis

Abstract

Spermiogenesis is the final stage of spermatogenesis, a differentiation process during which unpolarized spermatids undergo excessive remodeling that results in the formation of sperm. The actin cytoskeleton and associated actin-binding proteins play crucial roles during this process regulating organelle or vesicle delivery/segregation and forming unique testicular structures involved in spermatid remodeling. In addition, several myosin motor proteins including MYO6 generate force and movement during sperm differentiation. MYO6 is highly unusual as it moves towards the minus end of actin filaments in the opposite direction to other myosin motors. This specialized feature of MYO6 may explain the many proposed functions of this myosin in a wide array of cellular processes in animal cells, including endocytosis, secretion, stabilization of the Golgi complex, and regulation of actin dynamics. These diverse roles of MYO6 are mediated by a range of specialized cargo-adaptor proteins that link this myosin to distinct cellular compartments and processes. During sperm development in a number of different organisms, MYO6 carries out pivotal functions. In Drosophila, the MYO6 ortholog regulates actin reorganization during spermatid individualization and male KO flies are sterile. In C. elegans, the MYO6 ortholog mediates asymmetric segregation of cytosolic material and spermatid budding through cytokinesis, whereas in mice, this myosin regulates assembly of highly specialized actin-rich structures and formation of membrane compartments to allow the formation of fully differentiated sperm. In this review, we will present an overview and compare the diverse function of MYO6 in the specialized adaptations of spermiogenesis in flies, worms, and mammals.

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Histochemistry and Cell Biology (2021) 155:323–340
https://doi.org/10.1007/s00418-020-01954-x
REVIEW
Diverse functions ofmyosin VI inspermiogenesis
PrzemysławZakrzewski1,3· MartaLenartowska1,2· FolmaBuss3
Accepted: 2 December 2020 / Published online: 2 January 2021
© The Author(s) 2020
Abstract
Spermiogenesis is the final stage of spermatogenesis, a differentiation process during which unpolarized spermatids undergo
excessive remodeling that results in the formation of sperm. The actin cytoskeleton and associated actin-binding proteins
play crucial roles during this process regulating organelle or vesicle delivery/segregation and forming unique testicular
structures involved in spermatid remodeling. In addition, several myosin motor proteins including MYO6 generate force
and movement during sperm differentiation. MYO6 is highly unusual as it moves towards the minus end of actin filaments
in the opposite direction to other myosin motors. This specialized feature of MYO6 may explain the many proposed func-
tions of this myosin in a wide array of cellular processes in animal cells, including endocytosis, secretion, stabilization of
the Golgi complex, and regulation of actin dynamics. These diverse roles of MYO6 are mediated by a range of specialized
cargo-adaptor proteins that link this myosin to distinct cellular compartments and processes. During sperm development in
a number of different organisms, MYO6 carries out pivotal functions. InDrosophila,the MYO6 ortholog regulates actin
reorganization during spermatid individualization and male KO flies are sterile. In C. elegans, the MYO6 ortholog mediates
asymmetric segregation of cytosolic material and spermatid budding through cytokinesis, whereas in mice, this myosin regu-
lates assembly of highly specialized actin-rich structures and formation of membrane compartments to allow the formation
of fully differentiated sperm. In this review, we will present an overview and compare the diverse function of MYO6 in the
specialized adaptations of spermiogenesis in flies, worms, and mammals.
Keywords Actin· C. elegans· Drosophila· Mouse· Myosin VI· Spermiogenesis
Introduction
Sequential stages ofmammalian spermiogenesis
Spermiogenesis is the final stage of spermatogenesis, an
evolutionally conserved differentiation process that results
in the formation of mature spermatozoa. In mammals, sper-
miogenesis can be divided through microscopic observations
into four distinct phases: the Golgi, the cap, the acrosome,
and the maturation phase (Toshimori 2009); (Fig.1). During
the Golgi phase, proacrosomal vesicles (Fig.1, 1) derived
from the trans-Golgi network and the endocytic pathway
fuse to form the acrosomal vesicle (Fig.1, 2), which contains
the acrosomal granule (Fig.1, 3). The acrosomal vesicle
adheres through a cytoskeletal plate called the acroplax-
ome (Fig.1, 4) to the nuclear envelope. As demonstrated
in rodents, the acroplaxome consists of actin filaments and
the intermediate filament protein, SAK57 (spermatogenic
cell/sperm-associated keratin of molecular mass 57kDa),
which forms the marginal ring terminating the acroplaxome
and connecting, together with additional linker proteins, the
acrosome to the nuclear lamina (Kierszenbaum etal. 2003a).
During the cap phase, the acrosomal vesicle flattens out and
spreads over the spermatid nucleus to form a cap (Fig.1,
5); (Toshimori 2009). This process of acrosomal reshaping
and spermatid elongation is mediated by the flexible F-actin
scaffold of the acroplaxome (Fig.1, 6); (Kierszenbaum etal.
2003a, 2011). During the acrosomal phase, another tran-
sient structure, the manchette, is formed (Fig.1, 7), which
* Folma Buss
fb207@cam.ac.uk
1 Department ofCellular andMolecular Biology, Faculty
ofBiological andVeterinary Sciences, Nicolaus Copernicus
University inToruń, Torun, Poland
2 Centre forModern Interdisciplinary Technologies, Nicolaus
Copernicus University inToruń, Torun, Poland
3 Cambridge Institute forMedical Research, The Keith
Peters Building, University ofCambridge, Hills Road,
CambridgeCB20XY, UK
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324 Histochemistry and Cell Biology (2021) 155:323–340
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consists of a perinuclear ring, and contains actin filaments
and microtubules that extend into the elongating sperm tail
(Kierszenbaum etal. 2002, 2003a, 2007). The manchette is
involved in sperm head shaping and protein transport along
the cytoskeleton important for formation of the sperm fla-
gellum. Finally, the elongation of spermatids is completed
during the maturation phase, and most of the spermatid cyto-
plasm and organelles are discarded in the form of residual
bodies that are phagocytosed by Sertoli cells. Two additional
testis-specific structures are formed during spermiogenesis
in mammals—the apical ectoplasmic specialization (apical
ES); (Fig.1, 8) and the tubulobulbar complexes (TBCs);
(Fig.1, 9). The apical ES is a specialized actin-rich structure
formed between spermatids and Sertoli cells and is com-
posed of parallel actin bundles sandwiched between the sper-
matid plasma membrane and the Sertoli cell’s endoplasmic
reticulum (ER); (Toyama 1976; Russell 1977; Franke etal.
1978; Sun etal. 2011). The apical ES anchors developing
spermatids to the Sertoli cells, positions the spermatid head
regions in the correct orientation, and supports the spermatid
movement across the seminiferous tubule. TBCs are also
assembled at the spermatid–Sertoli cell interface and are
involved in the internalization of cell–cell junctions to facili-
tate sperm release (Upadhyay etal. 2012; Vogl etal. 2013).
TBCs consist of a long endocytic proximal tubule stabilized
and cuffed by a dense actin meshwork, an actin-free swollen
bulbular region surrounded by endoplasmic reticulum, and a
short distal tubule terminating in a clathrin-coated pit (Rus-
sell and Clermont, 1976; Russell 1979; Vogl etal. 1985).
After the reorganization of the apical ES and internaliza-
tion of cellular attachments via the TBCs, spermatogenesis
ends with sperm release to the lumen of seminiferous tubules
in a process called spermation. The still non-motile sperm
is transported to the epididymis, where the major part of
the maturation process occurs, before the final capacitation
to acquire hypermotility that takes place within the female
reproductive tract (Skerget etal. 2015). Moreover, some of
the luminal components of the epididymis, which are crucial
for final sperm maturation, such as the androgen binding
protein, transferrin, and immobilin, are also endocytosed and
recycled by the microvillar epididymal epithelium (Zhou
etal. 2018).
Actin cytoskeleton inspermiogenesis
Directly after meiosis, haploid spermatids enter spermio-
genesis as round unpolarized cells that undergo excessive
remodeling leading to the formation of mature sperm. In
mammals, the actin cytoskeleton plays an undisputed role at
several key points during this process serving as a cytoskel-
etal track to guide exocytic vesicles from the Golgi to the
acrosome or from the manchette to the centrosome/axoneme.
In addition, actin filaments are crucial for the assembly
and remodeling of testis-specific structures important for
spermatid development, including the acrosome–acroplax-
ome–manchette complex, the apical ES, and the TBCs (Lie
etal. 2010b; O’Donnell etal. 2011; Upadhyay etal. 2012;
Qian etal. 2014a, b; Dunleavy etal. 2019; Pleuger etal.,
2020; Yang and Yang 2020). Actin dynamics is spatiotem-
porally regulated by different actin-binding proteins (ABPs)
and some of these actin regulators have been shown to be
involved in mammalian spermiogenesis. Localization data in
Fig. 1 Schematic diagram highlighting the sequential steps of mouse
spermiogenesis. During the Golgi phase, proacrosomal vesicles (1)
fuse to form the acrosomal vesicle (2), which contains the acroso-
mal granule (3). The acrosomal vesicle adheres to the nuclear enve-
lope through the acroplaxome (4). During the cap phase, the acroso-
mal vesicle flattens and spreads over the nucleus to form a cap (5).
Acrosomal reshaping and spermatid elongation is mediated by the
acroplaxome (6). During the acrosomal phase, the manchette (7) par-
ticipates in the formation of the sperm flagellum. At this stage, sper-
matids are attached to Sertoli cells through the apical ES (8) which
also supports their movement across the seminiferous tubule. Finally,
during the maturation phase, the elongation of the spermatid is com-
pleted and most of the cytoplasm and organelles are removed and
phagocytosed by Sertoli cells. The TBCs form at the spermatid-Ser-
toli cell interface (9) and internalize cell–cell junctions supporting the
sperm release. Sc Sertoli cell, SpT spermatid
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325Histochemistry and Cell Biology (2021) 155:323–340
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rat testes are available for a number of ABPs, such as espin,
fimbrin, vinculin, plastin-3, and EPS8 (epidermal growth
factor receptor pathway substrate 8), that are present at the
apical ES (Grove and Vogl 1989; Grove etal. 1990; Bartles
etal. 1996; Chen etal. 1999; Lie etal. 2009; Siu etal. 2011;
Li etal. 2015) whereas the highly branched actin filament
network of TBCs has been shown to associate with differ-
ent actin regulators including cofilin, cortactin, N-WASP
(neuronal Wiskott-Aldrich syndrome protein), and ARP3
(actin-related protein 3) both in rats and mice (Guttman etal.
2004a; Young etal. 2009; Lie etal. 2010a). Furthermore,
cortactin regulates the acrosome–acroplaxome–manchette
complex dynamics and tyrosine phosphorylated cortactin
has been localized to the rat acroplaxome, while non-phos-
phorylated cortactin to the manchette (Kierszenbaum etal.
2008). Finally, a number of actin-based myosins are involved
at different stages of sperm development in mammals (Li
and Yang 2016). Myosin Va (MYO5a), for example, plays
a role in acrosome biogenesis in rodents by transporting
proacrosomal vesicles along actin filaments and facilitating
their fusion to the acroplaxome (Kierszenbaum etal. 2003b,
2004). Myosins of class V have also been suggested to be
involved in the intra-manchette transport for the delivery
of cargo to the centrosome in the developing sperm flagel-
lum during human spermiogenesis (Hayasaka etal. 2008).
Myosin VIIa (MYO7a) has been implicated in the adhesion
and transport of developing spermatids across the rat semi-
niferous epithelium and its depletion perturbs the spatiotem-
poral expression of different ABPs involved in spermiogen-
esis (Velichkova etal. 2002; Wen etal. 2019). Finally, gene
expression profiling in rodent tissues has revealed that tran-
scripts of the minus-directed myosin VI (MYO6) are present
in mouse testes (Avraham etal. 1995) and that two different
MYO6 isoforms are expressed in mouse and rat testes (Buss
etal. 2001; Zakrzewski etal. 2017).
Structure andfunction ofMYO6
Myosin motor proteins translocate along actin filaments,
form dynamic tethers between the actin cytoskeleton and
membrane compartments or regulate actin filament organi-
zation and dynamics. MYO6 is a highly unusual myosin
motor, which unlike other myosins moves towards the minus
end of actin filaments (Wells etal. 1999). This specialized
feature of MYO6 may explain the many proposed functions
of this myosin in a wide array of cellular processes in animal
cells. Overall, the structure of MYO6 follows the general
domain organization of other proteins in the myosin fam-
ily: an N-terminal motor domain (head) which binds actin
filaments and ATP and converts biochemical energy into
mechanical force, a neck region (lever arm) that binds light
chains or calmodulins, which regulate motor properties and
a tail domain important for dimerization and cargo-binding
and precise spatiotemporal targeting. In addition, MYO6 has
two unique inserts: the first one in the motor domain near
the ATP-binding pocket mayregulate ATPase activity, while
the second insert, also called the “reverse gear”, between
the motor domain and the lever arm is responsible for the
reverse reposition of the lever arm at the end of the power
stroke allowing its “backward” movement (Menetrey etal.
2005). Finally, the MYO6 tail is alternatively spliced at two
sites giving rise to four different tissue-specific MYO6 vari-
ants with either a large insert (LI, 12–32 amino acids) or a
small insert (SI, 9amino acids), no insert or both inserts
(LI + SI); (Buss etal. 2001; Dance etal. 2004; de Jonge etal.
2019). These inserts regulate the binding of different adaptor
proteins and thus determine the subcellular localization and
function of MYO6 in different cell types and tissues (Buss
etal. 2001; Wollscheid etal. 2016; O’Loughlin etal. 2018).
To date, MYO6 has been implicated in a number of cel-
lular processes including endocytosis, secretion, stabiliza-
tion of the Golgi complex, autophagy, mitophagy, regula-
tion of actin dynamics, myogenesis, and transcription (Buss
etal. 2001; Warner etal. 2003; Sahlender etal. 2005; Tum-
barello etal. 2012, 2013, 2015; Tomatis etal. 2013, 2017;
Karolczak etal. 2015b; Fili etal. 2017; Kruppa etal. 2018;
Majewski etal. 2018; O’Loughlin etal. 2018; de Jonge etal.
2019). One of the key factors that regulates the function of
MYO6 in these diverse processes is its ability to bind dif-
ferent adaptor proteins within the cargo-binding domain in
the tail. For instance, via interaction with the clathrin and
endocytic adaptor protein DAB2 (Disabled-2), the LI iso-
form of MYO6 is targeted to clathrin-coated pits/vesicles,
where it facilitates receptor uptake at the apical domain of
polarized epithelial cells (Morris etal. 2002; Wollscheid
etal. 2016). In contrast, the MYO6 NI isoform interacts with
GIPC1 (GAIP C-terminus-interacting protein) and TOM1/
L2 (Target of Myb protein 1/Target of Myb-like protein 2),
which target MYO6 to APPL1-(Adaptor Protein, Phosphoty-
rosine Interacting With PH Domain And Leucine Zipper 1)
and RAB5-(Ras-related in brain) positive early endosomes
and regulate translocation of these early endosomes through
the dense actin cortex below the plasma membrane, which
facilitates their maturation and regulates downstream endo-
somal signaling (Aschenbrenner etal. 2003; Tumbarello
etal. 2012, 2013; Masters etal. 2017; O’Loughlin etal.
2018; de Jonge etal. 2019). Further MYO6-binding pro-
teins are optineurin, NDP52 (nuclear dot protein 52kDa),
and TAX1BP (Tax1-binding protein 1), which are selective
autophagy receptors and are believed to link MYO6 func-
tion to autophagosome maturation (Sahlender etal. 2005;
Morriswood etal. 2007; Tumbarello etal. 2013, 2015). At
present, it is not known whether MYO6 operates in these
diverse cellular processes as a cargo transporter or as a
protein/organelle anchor. Finally, MYO6 has recently been
linked to several RhoGEF complexes, which suggests an
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326 Histochemistry and Cell Biology (2021) 155:323–340
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active role in modulating actin track dynamics as well as
in regulating septin organization. MYO6, for example, has
been identified in a complex with LRCH3 (leucin-rich repeat
and calponin homology domain-containing protein 3) and
DOCK7 (Dedicator of cytokinesis protein 7), a GEF for
RAC (Ras-related C3 botulinum toxin substrate) and CDC42
(cell division control protein 42 homolog), and in a complex
with GIPC1 and LARG (Leukemia-associated Rho guanine
nucleotide exchange factor), a RhoGEF (O’Loughlin etal.
2018; de Jonge etal. 2019).
The Snell’s waltzer (sv/sv) mouse contains a spontane-
ous mutation in the Myo6 gene, which leads to the complete
absence of MYO6 in the homozygous mouse (Avraham etal.
1995). Snell’s waltzer mice are deaf and exhibit a tail-chaser
phenotype due to vestibular dysfunction that results from the
neurosensory epithelium degeneration in the inner ear (Deol
and Green 1966; Avraham etal. 1995; Self etal. 1999; Roux
etal. 2009). These mice also display several other defects
in a variety of tissues and organs, such as aberrations in the
Golgi morphology, reduced secretion, defective endocytosis,
and impaired morphology of brush border enterocytes and
hippocampal neurons (Warner etal. 2003; Osterweil etal.
2005; Ameen and Apodaca 2007; Collaco etal. 2010; Gotoh
etal. 2010; Hegan etal. 2012, 2015b). Moreover, profound
fibrosis and both cardiac and pulmonary vascular endothe-
lial defects were observed in MYO6 mutant mice (Hegan
etal. 2015a). Interestingly, not only in vertebrates but also in
Drosophila depletion of the MYO6 ortholog, jaguar, leads to
a variety of abnormal phenotypes, especially during embryo-
genesis, and interestingly, spermiogenesis (Mermall etal.
1995; Deng etal. 1999; Hicks etal. 1999; Millo etal. 2004).
Diverse functions ofMYO6
inspermiogenesis
MYO6 inDrosophila spermatid maturation
At the final step ofDrosophilaspermatogenesis called sper-
matid individualization, a cyst of 64 syncytial spermatids
is reorganized into individual sperm by membrane remod-
eling and removal of cytoplasmic content (Tokuyasu etal.
1972; Noguchi and Miller 2003). This process is driven by
long-lived actin structures, so-called actin cones, which
assemble around spermatid nuclei and travel synchronously
along the axonemes to the ends of the tails (Fig.2). As they
move, most of the spermatid cytoplasm is pushed out of
the flagellum, accumulating in the cystic bulge, and finally
discarded in the form of a waste bag. At the same time,
the cyst membrane is reorganized into individual sperm
membranes (Fig.3c; arrow shows a membrane connecting
actin cones, which is then pulled over individual sperma-
tids). Interestingly, newly formed actin cones are composed
exclusively of actin bundles, whereas moving cones develop
two domains—a rear region of parallel bundles and a dense
actin meshwork at the front (Fig.3d, e); (Noguchi etal.
Fig. 2 Spermatid individualization in Drosophila. In each testicular
cyst a syncytial membrane is reorganized into individual membranes
encasing 64 spermatids during individualization. This process is
driven by actin cones, which assemble around nuclei of spermatids
and move synchronously down the length of the axonemes. Early
actin cones are made of parallel actin bundles and contain jaguar
(ortholog of MYO6; red dots). As they move, the actin cones separate
into two distinct domains—at the rear end parallel actin filament bun-
dles predominate, whereas at the front actin filaments form a dense
meshwork. At this stage, jaguar concentrates at the front of the actin
cones. While the cytoplasm and organelles are extruded from sper-
matids, the cystic bulge forms. Remnants of the trailing cytoplasm
can be observed between the moving actin cones. When the actin
cones reach the end of the cyst, excess membrane and cytoplasm are
pinched off in the form of the waste bag and the spermatids are left
completely encased in individual membranes and with fully formed
flagella. Red arrow shows the direction of movement of the actin
cones
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327Histochemistry and Cell Biology (2021) 155:323–340
1 3
2006, 2008). In wild-typeDrosophilatestes, at the begin-
ning of sperm individualization, jaguar, also called myo-
sin heavy chain 95F (encoded by jar) is present throughout
the actin cones, whereas later, when the individualization
cones move away from the spermatid nuclei, it concentrates
at the front of the actin filament meshwork (Fig.3a, b). In
jaguar-depleted (jar1/jar1) flies actin filament assembly is
disrupted causing loss of the actin meshwork at the front
of the cone and as a result the movement of the actin cones
stops before individualization is completed (Hicks etal.
1999; Rogat and Miller 2002; Noguchi etal. 2006; Lenar-
towska etal. 2012). Cellular components are no longer
Fig. 3 Localization of jaguar (MYO6 ortholog) and ultrastructure
of Drosophila actin cones. a Localization of jaguar (red) by immu-
nofluorescence in actin cones (green) at the beginning of spermatid
individualization. Jaguar is present throughout the actin cones (yel-
low indicates overlap between jaguar and actin) that form around
the nuclei of spermatids (blue). b Immunofluorescence localization
of jaguar (red) in actin cones (visualized in green) at a later stage of
spermatid individualization, when jaguar forms a dense band at the
front of the moving actin cones. c Ultrastructural analysis of actin
cones in a cyst isolated from Drosophila testis. Arrow indicates the
syncytial membrane, which progresses downwards to separate the
spermatids during individualization. d Ultrastructural visualization of
actin cones in the cystic bulge decorated by myosin-II subfragment 1,
which highlights the two distinct domains of the actin cones, a dense
actin meshwork at the front and parallel actin filaments at the rear.
The actin polymerization in the rear region drives cone movement
and the actin meshwork at the front ensures exclusion of the cyto-
plasm and reorganization of the syncytial membrane. e High resolu-
tion electron microscope image of a single actin cone decorated by
myosin-II subfragment 1. ac actin cone, ax axoneme, cy cytoplasm, ic
individualization complex, m mitochondrion, tcy trailing cytoplasm.
White arrow in a shows the direction of actin cones movement, which
is the same for b–e. Bars 5µm (a–b, d), 1µm (c, e)
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328 Histochemistry and Cell Biology (2021) 155:323–340
1 3
removed from mutant spermatids and sperm tails are not
always separated by individual plasma membranes. All these
cellular disruptions lead to the cessation of the individualiza-
tion process and male infertility, which highlights a crucial
role for jaguar during Drosophila spermiogenesis (Noguchi
etal. 2006).
Polarized distribution of selected ABPs in moving Dros-
ophila actin cones has also been demonstrated. The actin
nucleating complex, ARP2/3, and its activator, cortactin, are
strongly enriched in the dense actin meshwork at the front
of the cone, while two actin-bundling proteins, quail (a vil-
lin ortholog) and singed (a fascin ortholog), are localized
at the rear end of the cone (Rogat and Miller 2002). Inter-
estingly, when the ARP2/3 is absent, the cones still move
but meshwork formation is compromised, and membrane
reorganization and cytoplasmic exclusion are abnormal, and
individualization fails. In contrast, when profilin (a regula-
tor of actin assembly) is absent, bundle formation is greatly
reduced, the meshwork still forms but no movement occurs
(Noguchi etal. 2008). Thus, the two different cone domains
at the front and rear are differentially regulated and have dif-
ferent functions during spermatid individualization: the bun-
dles stabilized by actin cross-linkers at the rear are required
for the cone movement, whereas the actin meshwork formed
by ARP2/3 at the cone front is involved in removing the
cytoplasm from the sperm tails. Interestingly, in jar1/jar1
testes, the distribution of selected ABPs is disrupted and
the specific localization of MYO6 at the front of the cone
maintains its shape and size (Rogat and Miller 2002; Isaji
etal. 2011). These findings indicate that jaguar stabilizes
actin cone structure and plays an anchoring role during
Drosophila spermiogenesis by tethering different cargo/
membranes to actin filament structures. Although the pre-
cise molecular mechanism of jaguar function in this process
is still unknown, both the motor domain and cargo-binding
tail domain are required for intracellular targeting of jaguar
and dense meshwork formation at the front of the cone dur-
ing Drosophila spermiogenesis (Noguchi etal. 2006; Isaji
etal. 2011). The correct targeting and function of jaguar
require the conserved RRL and LWY motif responsible for
binding molecular partners as well as the WKA motif that
binds PtdIns(4,5)P2, which indicates that adaptor protein as
well as lipid binding are required for jaguar function at the
front of moving actin cones (Isaji etal. 2011). Unfortunately,
the jaguar adaptor proteins involved in this process in Dros-
ophila have not been identified so far.
Proposed function ofMYO6 inC. elegans spermatid
differentiation
Morphological rearrangements in nematode spermatogen-
esis do not lead to the formation of flagellated sperm, but
instead give rise to spermatozoa that use amoeboid motility.
In C. elegans, the differentiation of haploid spermatids into
mature spermatozoa involves the asymmetric segregation of
cellular material (Fig.4); (Kelleher etal. 2000). While mito-
chondria and specialized Golgi-derived organelles are sorted
into the developing sperm, other surplus organelles and
components, such as ribosomes, actin filaments and microtu-
bules, are removed from the spermatids and deposited in the
residual body (RB). This asymmetric division of cytosolic
content, the RB formation to collect the excluded material,
followed by spermatid release from the RB through cytoki-
nesis, involves two myosin motors, NMY2, the ortholog of
human non-muscle myosin II (NMII, encoded by nmy-2)
and spe-15, the ortholog of human MYO6 (encoded by spe-
15) (Kelleher etal. 2000; Hu etal. 2019a). NMY2 drives an
incomplete cytokinesis by forming an actomyosin ring that
initiates cleavage furrow ingression, however, fails to com-
plete restriction. The NMY2-mediated incomplete cytokine-
sis is believed to provide the force for RB expansion. Spe-
15, in contrast, assembles into an actin-spe-15 ring, which
constricts the membrane between the spermatid and RB and
finally causes spermatid budding through spe-15-dependent
cytokinesis. This process is dependent on the spe-15 adap-
tor protein GIPC (encoded in C. elegans by gipc-1), which
may indicate that dimerization/multimerization of MYO6 is
required for cytokinesis and spermatid release. Depletion of
NMY2 or spe-15 causes defects in the asymmetric segrega-
tion of cytosolic components.
MYO6 inmammalian spermiogenesis
Localization ofMYO6 inmouse testes
MYO6 is broadly expressed in different animal tissues
including the testes in humans, rodents, worms and Dros-
ophila (Kelleher etal. 2000; Kellerman and Miller 1992;
Hasson and Mooseker 1994; Avraham etal. 1995, 1997).
Moreover, PCR analysis demonstrated that two MYO6 iso-
forms (the SI and NI) are expressed in rodent testes (Buss
etal. 2001; Zakrzewski etal. 2017) and are associated with
several key actin-rich structures throughout sperm develop-
ment and maturation in mice (Figs.5 and 6); (Zakrzewski
etal. 2017, 2020a, b). During the Golgi phase, MYO6 is
present at/around the Golgi complex adjacent to the acro-
some-nuclear pole, including the trans-Golgi network and
uncoated and as well as coated vesicles and at the inner
acrosome membrane–acroplaxome interface (Fig.5a–d);
(Zakrzewski etal. 2017, 2020a). During the cap phase,
MYO6 continues to be present at the trans-Golgi network
and surrounding vesicles and at the acroplaxome, espe-
cially directly below the electron-dense acrosomal granule
(Fig.5e–h); (Zakrzewski etal. 2017, 2020a). During the
following phase, the acrosome or elongation phase, when the
acrosome spreads over the spermatid nucleus, MYO6 is still
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329Histochemistry and Cell Biology (2021) 155:323–340
1 3
present at the acroplaxome (Fig.6a–d); (Zakrzewski etal.
2017, 2020a). Finally, during the maturation phase, MYO6
is found at TBCs, predominantly at the bulbular region and
at an early endocytic compartment (Fig.6e–h); (Zakrzewski
etal. 2020b). In the following sections, we will describe how
the loss of MYO6 impacts on the organization of the actin
cytoskeleton and specialized membrane compartments dur-
ing the different stages of mammalian spermiogenesis in the
Snell’s waltzer mouse.
Loss ofMYO6 causes morphological changes
duringacrosome biogenesis inmouse
Defects in Golgi organization and vesicle trafficking Sev-
eral membrane trafficking routes have been proposed to be
involved in acrosome formation, including the transport
of proacrosomal vesicles from the trans-Golgi network to
the developing acrosome in the secretory pathway (Figs.1
and 5a), the direct transport from the plasma membrane
along the endocytic pathway or directly from lysosomes to
the acrosome (Clermont and Tang 1985; Toshimori 1998,
2009; Kierszenbaum etal. 2003a). Thus, acrosome biogen-
esis includes the dynamic flow of vesicles at the intersec-
tion of exocytic and endocytic membrane trafficking routes
(Raposo etal. 2007; Berruti etal. 2010; Berruti and Paiardi
2011; Delevoye etal. 2019). Indeed, the importance of the
secretory pathway in acrosome biogenesis is highlighted by
the finding that lack of GOPC (Golgi-associated PDZ- and
coiled-coil motif-containing protein), which is involved in
vesicle transport from the Golgi complex in other cell types,
inhibits acrosome formation in developing mouse sper-
matids (Yao et al. 2002). GOLGA3 (Golgin subfamily A
member 3) and PICK1 (protein interacting with C kinase
1), which both have been shown to interact with GOPC,
are also present at the Golgi complex in developing sper-
matids and thus may contribute to secretory vesicle forma-
tion and delivery during acrosome development (Bentson
etal. 2013; Xiao etal. 2009). Proacrosomal vesicles may
be transported via two different cytoskeletal routes: along
actin filaments, involving unconventional MYO5a or along
microtubules with the help of KIFC1 (Kinesin family mem-
ber C1); (Kierszenbaum etal. 2003b, 2004, 2011; Yang and
Sperry 2003). In addition, our recent results have shown that
also MYO6 plays a role during the early stages of acrosome
biogenesis. Ultrastructural analysis of developing Snell’s
waltzer spermatids revealed several defects affecting acro-
some formation, including partial disruption of the Golgi
complex and impairment of proacrosomal vesicular traffick-
Fig. 4 Model of spe-15 (MYO6
ortholog) function during
spermatid differentiation in C.
elegans. After meiosis, two
haploid spermatids remain
connected and differentiate by
shedding residual cytoplasm
in the form of residual body.
Spermatid budding is mediated
by spe-15 (red). Mitochondria
are transported to spermatids,
whereas all ribosomes and
remaining organelles are packed
to the residual body. Spermatids
detach from the residual body
following the cytokinesis medi-
ated by spe-15 and next mature
into spermatozoa (based on Hu
etal. 2019a, b)
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330 Histochemistry and Cell Biology (2021) 155:323–340
1 3
ing (Zakrzewski etal. 2020a). A more detailed analysis of
the Golgi morphology insv/svspermatids demonstrated dif-
ferent morphological phenotypes—the asymmetrical orien-
tation of the Golgi complex in the relation to the upper pole
of spermatid nucleus and the loss of Golgi integrity(Zakrze-
wski etal. 2020a). Enlarged and swollen Golgi stacks and
vesicles were observed insv/svspermatids, similar to those
seen in cells treated with filamentous-actin-depolymerizing
agents (Egea et al. 2013, 2015). Interestingly, MYO6 is
present at the Golgi complex in different cell types and its
depletion has been shown to cause changes in Golgi mor-
phology, including its fragmentation, elongation, and reduc-
tion in size (Buss etal. 1998; Warner etal. 2003; Sahlender
etal. 2005; Puri etal. 2010; Majewski etal. 2011; Karolczak
Fig. 5 Localization of MYO6 in mouse developing spermatids dur-
ing the Golgi and cap phases. a During the Golgi phase, MYO6 (red
dots) localizes to the trans-Golgi network, proacrosomal vesicles and
acroplaxome below the acrosomal granule. b, c Immunofluorescence
localization of MYO6 (red) at the Golgi complex (b) and at the acro-
plaxome (green, c). Arrowhead indicates the Golgi complex in (b)
and the area below the acrosomal granule in (c). d Ultrastructural
localization of MYO6 using immunogold labeling at the trans-Golgi
network and on the surface of the acrosomal vesicles (arrows). e Dur-
ing the cap phase, MYO6 (red dots) localizes to the trans-Golgi net-
work, proacrosomal vesicles and acroplaxome below the acrosomal
granule. f and g Immunofluorescence localization of MYO6 (red) at
the acroplaxome (green). Arrowheads indicate area below the acro-
somal granule. h Ultrastructural localization of MYO6 using immu-
nogold labeling at the acroplaxome below the acrosomal granule
(arrows). Panel d is modified from Zakrzewski et al. (2017) (pub-
lished under CC BY 4.0). af actin filament, ag acrosomal granule, av
acrosomal vesicle, ax acroplaxome, cy cytoplasm, m mitochondrion,
n nucleus, pav proacrosomal vesicles, trans-G trans-Golgi network,
Sc Sertoli cell, SpT spermatid. Bars 1µm
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331Histochemistry and Cell Biology (2021) 155:323–340
1 3
etal. 2015a). Therefore, it is tempting to speculate that also
in mouse testes MYO6 may play an anchoring role during
spermiogenesis linking Golgi membranes to the surround-
ing actin filaments to maintain its morphology and position
close to the developing acrosome.
The two splice variants of MYO6, the NI and SI isoforms,
which are both expressed in mammalian testes, are not only
important for exocytosis and the transport and tethering of
secretory vesicles but the NI isoform of MYO6 is also pre-
sent at early endosomes, where it is involved in the early
stages of endocytosis (Buss etal. 2001; Aschenbrenner etal.
2003; Dance etal. 2004; Au etal. 2007; Chibalina etal.
2007; Inoue etal. 2008; Puri 2009, 2010; Majewski etal.
2010; Bond etal. 2012; Tumbarello etal. 2012; Tomatis
etal. 2013) Therefore, it is tempting to hypothesize that
MYO6 may participate in the transport of exocytic vesicles
from the Golgi complex and/or endocytic vesicles to the
developing acrosomal vesicle.
At present, however, we have very little insight into the
exact function of MYO6 during acrosome biogenesis. MYO6
Fig. 6 Localization of MYO6 in mouse developing spermatids during
the acrosome and maturation phases. a During the acrosome phase,
MYO6 (red dots) localizes to the acroplaxome below the acrosomal
granule. b, c Immunofluorescence localization of MYO6 (red) at
the acroplaxome (actin visualized in green) in elongating sperma-
tids during the acrosome phase. Arrowheads indicate area below the
acrosomal granule. d Ultrastructural localization of MYO6 using
immunogold labeling at the acroplaxome below the acrosomal gran-
ule and the acrosome (arrows). Panel d is modified from Zakrzewski
etal. (2017) (published under CC BY 4.0). e During the maturation
phase, MYO6 (red dots) is concentrated at the bulbs of the TBCs
and APPL1-positive early endosomes. f Immunofluorescence locali-
zation of MYO6 (red) at the spermatid-Sertoli cell interface in the
seminiferous epithelium on a semi-thin paraffin section. During this
stage, maturing spermatids are close to the lumen of the seminifer-
eous tubules. g Immunofluorescence localization of MYO6 (red) in
the endocytic compartment of the TBCs (actin visualized in green).
h Ultrastructural localization of MYO6 using immunogold label-
ling in early endosomes in a spermatid during the maturation phase
(arrows). ac acrosome, af actin filament, ag acrosomal granule,
APPL1 + APPL1-positive early endocytic vesicle, av acrosomal vesi-
cle, ax acroplaxome, EEA1 + EEA1-positive early endosome, eev
early endocytic vesicle, er endoplasmic reticulum, es apical ES, lst
lumen of seminiferous tubule, n nucleus, Sc Sertoli cell, se seminifer-
ous epithelium, SpT spermatid, tbc tubulobulbar complex. Bars 5µm
(f), 1µm (b–d, g), 500nm (h)
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332 Histochemistry and Cell Biology (2021) 155:323–340
1 3
could be involved in short-range transport of proacrosomal
vesicles in the opposite direction to MYO5a. Alternatively,
MYO6 could be involved in the tethering of proacrosomal
vesicles to the surrounding actin cytoskeleton. In mouse
spermatids, during acrosome biogenesis, the MYO6-binding
partner TOM1/L2 is present on vesicular structures located
between the trans-Golgi network and maturing acrosome
(Zakrzewski etal. 2020a). TOM1/L2 is a monomeric pro-
tein, which forms a 1:1 complex with MYO6 and does not
induce dimerization or multimerization of MYO6 in con-
trast to other binding partners, such as DAB2 or GIPC1 (Yu
etal. 2009; Shang etal. 2017; Hu etal. 2019b). Thus, the
monomeric MYO6 in complex with TOM1/L2 may perform
a tethering function, in contrast to a dimeric/multimeric
myosin, which can move processively over short distances.
Although the exact cellular function of the MYO6-TOM1/
L2 complex remains to be established, both proteins are
present on early endosomes and facilitate the delivery of
these endosomes to autophagosomes required for their
maturation and fusion with lysosomes (Tumbarello etal.
2012; O’Loughlin etal. 2018; de Jonge etal. 2019). Indeed,
another observation in Snell’s waltzer spermatids suggests
that the absence of MYO6 may affect the fusion of proacro-
somal vesicles with the developing acrosome, as the number
of proacrosomal vesicles appears to be elevated (Zakrzewski
etal. 2020a). A similar phenotype was observed in mice
depleted of ATG7 (autophagy-related protein 7), which high-
lights a potential involvement of the autophagy pathway in
acrosome biogenesis (Wang etal. 2014). These observations
support the notion that in mouse testes, the MYO6-TOM1/
L2 complex may support tethering of proacrosomal vesicles
in the vicinity of the upper pole of the spermatid nucleus to
facilitate fusion between the proacrosomal vesicles and the
developing acrosome (Zakrzewski etal. 2020a). However,
further research is required to establish the exact function
of MYO6 during acrosome biogenesis in mammals and to
determine the polarity of actin filaments around proacroso-
mal vesicles, the Golgi complex, and the acrosome, which
directs the minus-end-directed movement of MYO6.
Loss of acrosome symmetry The fusion of proacrosomal
vesicles leads to the formation of the glycoprotein-rich acro-
somal vesicle, which gradually spreads over the spermatid
nucleus forming a cap (Figs.1 and 5a, e); (Toshimori etal.
2009). Both proacrosomal and acrosomal vesicles contain an
electron-dense core with an amyloidogenic structure called
the acrosomal granule, which is enriched in acidic hydro-
lytic enzymes (Khawar et al. 2019). This granule closely
associates with the inner acrosomal membrane at the center
of the acroplaxome. As the acrosomal vesicle flattens, the
electron-dense material of the granule fills the entire acro-
somal matrix (Toshimori etal. 2009). Similar to dense-core
secretory granules, the low intraluminal pH of the acroso-
mal vesicle may drive the concentration and compartmen-
talization of acrosomal proteins into the acrosomal granule
(Moreno etal. 2000). Throughout acrosome biogenesis, the
acrosomal granule is tethered at the center of the acrosomal
vesicle. However, very little is known about the possible
mechanisms that anchor and maintain the central position
of this granule. Interestingly, our recent results have shown
that in MYO6-deficient spermatids, the central localization
of acrosomal granules is lost in almost a quarter of Snell’s
waltzer spermatids (Zakrzewski etal. 2020a). In some of
the cells, the granule is completely detached from the inner
acrosomal membrane and appears to “float” freely inside the
acrosomal vesicle or is even absent. Similar anomalies have
been observed in other mouse mutants that lack for example
the expression of ZPBP1 (Zona pellucida binding protein
1); (Lin etal. 2007). More severe deformities, such as acro-
somal granules, attached ectopically to the outer acrosomal
membrane have been described in Dpy19l2−/− (probable
C-mannosyltransferase-null) male mice (Pierre etal. 2012).
Uneven distribution of acrosomal material has also been
noted in sperm populations lacking the expression of pro-
protein convertase subtilisin/kexin type 4 (PCSK4); (Tar-
dif etal. 2012). Finally, acrosome malformations have also
been reported in Acrbp−/− (acrosin-binding protein-defi-
cient) male mice completely lacking an acrosomal granule
(Kanemori etal. 2016). The acrosomal defects reported for
these mouse mutants may be caused by the destabilization
of the multi-layered structure of the acrosome, the improper
compaction of the acrosome or the altered processing of the
acrosomal proteins (Lin etal. 2007; Pierre etal. 2012; Tar-
dif etal. 2012; Kanemori etal. 2016).
At present, we can only speculate what causes the dock-
ing defect of the acrosomal granule in Snell’s waltzer mice
as acrosome asymmetry is the only significant change linked
to acrosome development in MYO6-deficient spermatids
(Zakrzewski etal. 2020a). MYO6 together with its binding
partner TOM1/L2 localizes to the acroplaxome right below
the acrosomal granule (Zakrzewski etal. 2020a). In MYO6-
deficient spermatids, TOM1/L2 is still present at the acro-
plaxome, which may suggest that MYO6 is not required for
the localization of TOM1/L2, but is the essential factor that
determines the correct localization of the acrosomal granule
within the acrosome. Although the molecular details of how
the MYO6-TOM1/L2 complex maintains the symmetrical
localization of the acrosomal granule during acrosome bio-
genesis are unknown, MYO6 may bind via its motor domain
to actin filaments in the acroplaxome and through the tail
domain to TOM1/L2 (Zakrzewski etal. 2020a). At the same
time, TOM1/L2 may interact with a transmembrane protein
of the inner acrosome membrane, which in turn may bind
with its luminal domain to proteins of the acrosomal granule.
This would provide a mechanism for docking of the acro-
somal vesicles across the inner acrosomal membrane and a
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333Histochemistry and Cell Biology (2021) 155:323–340
1 3
TOM1/L2-MYO6 complex to the underlying actin filaments
of the acroplaxome. While the TOM1/L2 interactome has
not been established in mouse testes, the three helices in the
core of the GAT domain of TOM1/L2 bind to ubiquitin and
thus could interact with ubiquitinated transmembrane recep-
tors in the inner acrosome membrane (Wang etal. 2010).
Indeed, ubiquitinated proteins have been detected at the
acrosomal granule, the inner acrosomal membrane and the
acroplaxome, which would support our proposed mechanism
of granule docking via the GAT domain of TOM1/L2 and
MYO6 binding to actin filaments within the acroplaxome
(Haraguchi etal. 2004; Rivkin etal. 2009; Nakamura etal.
2013; Zakrzewski etal. 2020a).
Absence ofMYO6 affects machinery involved insperm
release inmouse
Defects in the spatial organization ofendocytic compart-
ment During spermiogenesis, the maturing spermatids
adhere to Sertoli cells and move across the seminiferous epi-
thelium with the help of the apical ES, a unique actin-based
and ER-associated anchoring junction (Figs.1 and 6a). To
allow the release of mature spermatozoa into the seminifer-
ous tubules, the apical ES is disassembled and internalized
(Adams et al. 2018). A second actin-rich structure is the
TBC, which projects from the developing spermatids into
the adjacent Sertoli cell cytoplasm and is believed to facili-
tate the internalization of the apical ES (Figs.1 and 6e). The
structure and molecular composition of TBCs suggests that
they are evolutionarily related to endocytic uptake pathways
involving clathrin. Indeed, clathrin-coated pits initiate the
formation of TBCs and remain at the tip of the extending
complexes (Russell and Clermont 1976). Furthermore, the
long tubular extensions of TBCs are associated with mem-
brane curvature sensing proteins, such as amphiphysin and
dynamin, which facilitates vesicle scission (Kusumi et al.
2007; Vaid etal. 2007). The overall structure of the TBC
and the narrow diameter of the tubular extensions is sup-
ported by a cuff of dense actin meshwork and several ABPs
(Sriram et al. 2016). The bulbar regions of the TBCs and
endocytic vesicles internalized from this region are associ-
ated with the early endosome marker RAB5 (Young et al.
2012; Adams and Vogl 2017). These endosomes also known
as sorting endosomes become positive for EEA1 (early
endosome antigen 1) and later recruit LAMP1 (lysosome-
associated membrane glycoprotein 1); (Guttman et al.
2004b; Du etal. 2013; Adams and Vogl 2017). Interestingly,
our recent results highlight that also MYO6 is localized at
the bulbar regions and to vesicles in close proximity to the
TBCs (Fig.6g, h); (Zakrzewski etal. 2020b). Furthermore,
we identified two MYO6 adaptor proteins, TOM1/L2 and
GIPC1, in a vesicular compartment at the TBCs. These vesi-
cles are positive for APPL1 (adapter protein containing PH
domain, PTB domain and leucine zipper motif 1), an adap-
tor protein present on a subset of RAB5-positive endosomes
that are negative for EEA1. It is well established in different
cell types and tissues that MYO6 localizes to and tethers
RAB5- and APPL1-positive early endosomes to the actin
cortex underneath the plasma membrane (Tumbarello etal.
2013; Masters etal. 2017; O’Loughlin etal. 2018). MYO6
maintains the localization of these APPL1-endosomes in the
cell periphery, which facilitates maturation and downstream
signaling events that precedes cargo processing in EEA1-
positive early/sorting endosomes (Tumbarello et al. 2013;
Masters etal. 2017). In testes, the lack of MYO6 expression
causes the disorganization of the TBCs and loss of spatial
integrity of the APPL1-positive early endosomal compart-
ment (Zakrzewski etal. 2020b). These results suggest that
MYO6 may stabilize the functional structure of the TBCs by
linking TOM1/L2-positive membranes to surrounding actin
filaments. After endocytosis from the bulbular region of the
TBC, MYO6 tethers and maintains the spatial position of
this TOM1/L2-GIPC1 and APPL1-positive endocytic clus-
ter, thus potentially enabling the maturation of this subset
of APPL1-containing early endosomes into EEA1-positive
endosomes. However, it is still not known whether the spa-
tial integrity of the TBCs is crucial for their function and
whether loss of the spatial arrangement of this compartment
is directly linked to the slightly reduced number of epididy-
mal sperm and reduced fertility in Snell’s waltzer males
(Zakrzewski etal. 2020b). The endocytosis of the junctional
protein nectin-3, for example, which forms heterotypic
intracellular adhesion junctions at the spermatid/Sertoli
cell interface (Rikitake etal. 2012; Adams and Vogl 2017)
is not completely blocked in sv/sv spermatids. Nectin-3 is
still endocytosed and present in vesicular structures in the
cytoplasm of sv/sv Sertoli cells; however, these vesicles are
dispersed and no longer concentrated where the TBCs are
clustering (Zakrzewski et al. 2020b). Thus, although the
endocytosis of junctional complexes is not entirely inhib-
ited, the dispersion of the TBC endocytic compartment may
affect the efficiency of forward trafficking and recycling of
nectin-3 to newly formed intercellular attachments in other
parts of the Sertoli cell.
Finally, MYO6 is also present together with cortactin
and ARP3 at the actin cuffs surrounding the long, extended
neck of the TBCs (Zakrzewski etal. 2020b). Interestingly, in
Snell’s waltzer mice, these two ABPs are no longer associ-
ated with this region of the TBCs, which may suggest a role
for MYO6 in the dynamic organization of the actin mesh-
work surrounding the long proximal tubules of the TBCs.
In Drosophila, the MYO6 ortholog, jaguar is required for
organization of actin cones during spermatid individualiza-
tion and both ABP, cortactin and ARP3 are displaced from
the front of the cones in jar1/jar1 flies (Rogat and Miller
2002; Noguchi etal. 2006). Although the exact role of jaguar
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334 Histochemistry and Cell Biology (2021) 155:323–340
1 3
in Drosophila spermatid individualization is still not known,
it seems to facilitate recruitment of different actin regula-
tors to the front of the actin cones, thereby, regulating actin
branching through recruitment of the ARP2/3 complex. In
mammals, MYO6 has been shown to form a complex with
LARG, a RhoGEF that modulates actin organization around
endosomes (O’Loughlin etal. 2018) and with DOCK7, also
a RhoGEF, which not only regulates actin dynamics but
also the assembly of septins structures along actin filaments
(Majewski etal. 2012; Sobczak etal. 2016; O’Loughlin etal.
2018). Thus, MYO6 may regulate actin polymerization and
organization around the TBCs by interacting with different
RhoGEFs.
Defects inthestructure oftheapical ES The apical ES not
only maintains the close contact between maturing sper-
matids and Sertoli cells but also enables the migration of
spermatids across seminiferous epithelium during sper-
miogenesis and contributes to the positioning of sperma-
tids with their flagella pointed towards the lumen of semi-
niferous tubules (Dunleavy et al. 2019). The apical ES is
a unique anchoring junction consisting of adherens junc-
tions (cadherins/catenins and nectin/afadin complexes),
tight junctions (JAM-C and CAR molecules) and focal
contacts (α6β1-integrin/laminin α3β3γ3 complex); (Wong
etal. 2007; Yan etal. 2008; Kopera etal. 2010). As sper-
miogenesis progresses and the maturing spermatids move
towards the apical compartment of the epithelium, the api-
cal ES undergoes rapid cycles of assembly and disassembly
facilitated by the tightly regulated reorganization of actin
filaments switching between a bundled and branched con-
figuration. Actin dynamics in the apical ES is regulated by
a number of ABPs including for example actin-bundling
proteins, such as EPS8, paladin and α-actinin, and proteins
regulating branched actin filament assembly, such as the
ARP2/3 complex, N-WASP, drebin E and filamin A (Young
etal. 2009; Li etal. 2011; Su etal. 2012; Qian etal. 2014a,b;
Xiao et al. 2014). Interestingly, the same actin nucleating
proteins that control effective actin reorganization in Dros-
ophila spermatid individualization—the ARP2/3 complex
and cortactin—are also involved in apical ES remodeling
(Chapin etal. 2001; Anahara etal. 2006; Lie etal. 2010a).
Our analysis using electron microscopy has shown that
MYO6 is also present at apical ES during sperm develop-
ment in mice (Zakrzewski etal. 2017). The signal for MYO6
was predominantly detected in the F-actin bundles of the
apical ES that surround the apical pole of the elongating
nuclei (Fig. 7a–d). Here, MYO6 may maintain the struc-
tural integrity of the apical ES by controlling actin filament
dynamics, similar to its role during Drosophila spermatid
maturation and in other mammalian cell types and tissues
(O’Loughlin et al. 2018). Indeed, our preliminary results
highlight structural defects, such as swollen ER cisternae
and local detachment of the actin bundles from the sper-
matid head, within the apical ES in MYO6-deficient males
(Fig. 8a, b). Interestingly, we did not observe any signifi-
cant morphological defects in sv/sv sperm (except for their
slightly reduced number), which would suggest the prema-
ture release of round/elongated spermatids to the lumen of
seminiferous tubules (Wen et al. 2019; Zakrzewski et al.
2020a). In addition to MYO6, also MYO7a and its bind-
ing partner KEAP1 (Kelch-like ECH-associated protein 1),
both localize to actin filament bundles within the ES com-
partment in Sertoli cells. Although MYO7a has been sug-
gested to play a role in spermatid and organelle transport
and adhesion during spermatogenesis, the MYO7a KO male
mice show no obvious structural disruptions of the apical
ES (Hasson etal. 1997; Velichkova etal. 2002; Wen etal.
2019). The knockdown of MYO7a in rat Sertoli cells or in
rat testes, however, induced severe disorganization of the
actin cytoskeleton across the seminiferous epithelium and
abnormal expression of selected ABPs (Wen etal. 2019).
Sertoli cell–spermatid adhesion and transport of organelles,
such as residual bodies and phagosomes, across the epithe-
Fig. 7 MYO6 localizes to the apical ES. During the maturation phase, MYO6 (red) is also present at the apical ES, where it localizes to actin
bundles (visualized in b and d in green) that enclose the maturing spermatids. Sc Sertoli cell,SpT spermatid.Bars 1µm
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335Histochemistry and Cell Biology (2021) 155:323–340
1 3
lium were grossly disrupted in MYO7a-deficient rat testes,
supporting a function for MYO7a in intracellular transport
and adhesion during spermiogenesis.
Perspectives andconcluding remarks
The unique reverse directionality of MYO6 may explain why
the activity of this motor is necessary at different stages of
spermiogenesis and its absence causes a set of specific phe-
notypes in mice. During the Golgi phase, MYO6 appears to
maintain the spatial organization of the Golgi complex and to
facilitate a short-distance transport and fusion of proacroso-
mal vesicles. During the following cap and acrosome phases,
depletion of MYO6 impacts on the correct localization of
the acrosomal granule and symmetry of developing acro-
some. Finally, during the maturation phase, loss of MYO6
disturbs the spatial integrity of the endocytic compartment at
the TBC. In addition, our preliminary observations suggest
that MYO6 may regulate actin dynamics linked to the apical
ES organization. Overall, our observations suggest that in
mouse testes, MYO6 plays a structural role during sperma-
tid development by regulating actin dynamics and anchor-
ing different membranous organelles to the surrounding
actin cytoskeleton. Although there are certain parallels in
the function of MYO6 and its orthologs in regulating actin
filament dynamics during Drosophila spermatid individu-
alization, C. elegans spermatid differentiation, and mouse
spermiogenesis, MYO6-deficiency in mouse spermatids is
less pronounced and the Snell’s waltzer male mice are only
sub-fertile, while MYO6-deficient flies and worms are ster-
ile (Kelleher etal. 2000; Noguchi etal. 2006; Zakrzewski
etal. 2020b). Although MYO6 is the only myosin known
so far that moves to the minus end of actin filaments, in the
opposite direction to all other myosins, it seems surprising
that the many different phenotypes uncovered at different
stages of spermiogenesis only cause a minor reduction in
male mouse fertility of the Snell’s waltzer mice. MYO6,
similar to MYO5a and MYO7a, appears to play highly spe-
cialized roles at distinct steps of murine spermatid devel-
opment; however, the lack of single parts of this complex
machinery does not completely disrupt the process of mouse
spermiogenesis.
While the role of the actin cytoskeleton in mammalian
spermiogenesis is a rapidly developing field, our current
understanding and recognition of the involvement of dif-
ferent myosins in this process is still less well documented.
As highlighted throughout this review, several questions
remain, especially in relation to the actin-based origins of
acrosome symmetry, regulation of TBC organization and
endocytosis, and function of the apical ES in the adhesion of
spermatids to Sertoli cells and spermiation. At present, the
suggested functions of MYO6 during mouse spermiogenesis
are predominantly based on morphological studies of fixed
cells and tissues. Although the functional analysis of mam-
malian spermiogenesis is very complex, future studies may
focus on identifying the wider MYO6 interactome in mam-
malian testis. This would allow us to understand which bind-
ing partners and cargo adaptor proteins regulate its function
and recruitment to different testicular compartments during
sperm development. Moving forward, the use of invitro Ser-
toli cell cultures and a combination of CRISPR/Cas9 KO
cells with super-resolution confocal microscopy may provide
further data to verify the proposed mechanisms of MYO6
function at different phases of spermiogenesis in mammals.
Author contributions PZ prepared the figures, acquired microscopic
images and wrote the paper. ML acquired microscopic images and
wrote the paper. FB wrote the paper, revised the text and figures.
Funding This project was supported by PRELUDIUM grant from
the National Science Centre (Poland) to PZ (2017/25/N/NZ3/00487),
Fig. 8 Loss of MYO6 causes ultrastructural disruptions of the apical
ES in maturing spermatids. Ultrastructural analysis of the apical ES
of a MYO6-expressing spermatid (a) and MYO6-deficient spermatid
(b). In the absence of MYO6 the apical ES appears to be disrupted
and detachment of the spermatid head from the apical ES and swell-
ing of the ER can be observed (arrow) (b). er endoplasmic reticulum,
mt manchette, n nucleus, Sc Sertoli cell. Bars 1µm
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

336 Histochemistry and Cell Biology (2021) 155:323–340
1 3
a ETIUDA doctoral scholarship from the National Science Centre
(Poland) to PZ (2018/28/T/NZ3/00002), a travelling fellowship funded
by The Company of Biologists to PZ (JCSTF-171105 (to P.Z.), and a
Medical Research Council grant to FB (MR/K000888/1). CIMR is
supported by the Wellcome Trust with a strategic award (100140) and
an equipment grant (093026).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
References
Adams A, Vogl AW (2017) High resolution localization of Rab5,
EEA1, and nectin-3 to tubulobulbar complexes in the rat testis.
Anat Rec (Hoboken) 300:1160–1170
Adams A, Sriram A, Vogl AW (2018) Internalization of intact inter-
cellular junctions in the testis by clathrin/actin-mediated endo-
cytic structures: tubulobulbar complexes. Anat Rec (Hoboken)
301:2080–2085
Ameen N, Apodaca G (2007) Defective CFTR apical endocytosis and
enterocyte brush border in myosin VI-deficient mice. Traffic
8:998–1006
Anahara R, Toyama Y, Maekawa M, Kai M, Ishino F, Toshimori K,
Mori C (2006) Flutamide depresses expression of cortactin in the
ectoplasmic specialization between the Sertoli cells and sperma-
tids in the mouse testis. Food Chem Toxic 44:1050–1056
Aschenbrenner L, Lee T, Hasson T (2003) Myo6 facilitates the trans-
location of endocytic vesicles from cell peripheries. Mol Biol
Cell 14:2728–2743
Au JS, Puri C, Ihrke G, Kendrick-Jones J, Buss F (2007) Myosin VI is
required for sorting of AP-1B-dependent cargo to the basolateral
domain in polarized MDCK cells. J Cell Biol 177:103–114
Avraham KB, Hasson T, Steel KP, Kingsley DM, Russell LB,
Mooseker MS, Copeland NG, Jenkins NA (1995) The mouse
Snell’s waltzer deafness gene encodes an unconventional myosin
required for structural integrity of inner ear hair cells. Nat Genet
11:369–375
Avraham KB, Hasson T, Sobe T, Balsara B, Testa JR, Skvorak AB,
Morton CC, Copeland NG, Jenkins NA (1997) Characteriza-
tion of unconventional MYO6, the human homologue of the gene
responsible for deafness in Snell’s waltzer mice. Hum Mol Genet
6:1225–1231
Bartles JR, Wierda A, Zheng L (1996) Identification and characteriza-
tion of espin, an actin-binding protein localized to the F-actin-
rich junctional plaques of Sertoli cell ectoplasmic specialization.
J Cell Sci 109:1229–1239
Bentson LF, Agbor VA, Agbor LN, Lopez AC, Nfonsam LE, Born-
stein SS, Handel MA, Linder CC (2013) New point mutation in
Golga3 causes multiple defects in spermatogenesis. Andrology
1:440–450
Berruti G, Paiardi C (2011) Acrosome biogenesis: revisiting old ques-
tions to yield new insights. Spermatogenesis 1:95–98
Berruti G, Ripolone M, Ceriani M (2010) USP8, a regulator of endo-
somal sorting, is involved in mouse acrosome biogenesis through
interaction with the spermatid ESCRT-0 complex and microtu-
bules. Biol Reprod 82:930–939
Bond LM, Arden SD, Kendrick-Jones J, Buss F, Sellers JR (2012)
Dynamic exchange of myosin VI on endocytic structures. J Biol
Chem 287:38637–38646
Buss F, Kendrick-Jones J, Lionne C, Knight AE, Cote GP, Luzio JP
(1998) The localization of myosin VI at the Golgi complex
and leading edge of fibroblasts and its phosphorylation and
recruitment into membrane ruffles of A431 cells after growth
factor stimulation. J Cell Biol 143:1535–1545
Buss F, Arden SD, Lindsay M, Luzio JP, Kendrick-Jones J (2001)
Myosin VI isoform localized to clathrin-coated vesicles with a
role in clathrin-mediated endocytosis. EMBO J 20:3676–3684
Chapin RE, Wine RN, Harris WM, Borchers CH, Haseman JK
(2001) Structure and control of a cell-cell adhesion complex
associated with spermiation in rat seminiferous epithelium. J
Androl 22:1030–1052
Chen B, Li A, Wang D, Zheng L, Bartles JR (1999) Espin con-
tains an additional actin-binding site in its N terminus and is
a major actin-bundling protein of the Sertoli cell-spermatid
ectoplasmic specialization junctional plaque. Mol Biol Cell
10:4327–4339
Chibalina MV, Seaman MNJ, Miller CC, Kendrick-Jones J, Buss F
(2007) Myosin VI and its interacting protein LMTK2 regulate
tubule formation and transport to the endocytic recycling com-
partment. J Cell Sci 120:4278–4288
Clermont Y, Tang XM (1985) Glycoprotein synthesis in the Golgi
apparatus of spermatids during spermiogenesis of the rat. Anat
Rec 213:33–43
Collaco A, Jakab R, Hegan P, Mooseker M, Ameen N (2010) Alpha-
AP-2 directs myosin VI-dependent endocytosis of cystic fibrosis
transmembrane conductance regulator chloride channels in the
intestine. J Biol Chem 285:17177–17187
Dance AL, Miller M, Seragaki S, Aryal P, White B, Aschenbrenner L,
Hasson T (2004) Regulation of myosin-VI targeting to endocytic
compartments. Traffic 5:798–813
de Jonge JJ, Batters C, O’Loughlin T, Arden SD, Buss F (2019) The
MYO6 interactome: selective motor-cargo complexes for diverse
cellular processes. FEBS Lett 593:1494–1507
Delevoye C, Marks MS, Raposo G (2019) Lysosome-related organelles
as functional adaptations of the endolysosomal system. Curr
Opin Cell Biol 59:147–158
Deng W, Leaper K, Bownes M (1999) A targeted gene silencing tech-
nique shows that Drosophila myosin VI is required for egg cham-
ber and imaginal disc morphogenesis. J Cell Sci 112:3677–3690
Deol MS, Green MC (1966) Snell’s waltzer, a new mutation affecting
behaviour and the inner ear in the mouse. Genet Res 8:339–345
Du M, Young J, De Asis M, Cipollone J, Roskelley C, Takai Y, Nicholls
PK, Stanton PG, Deng W, Finlay BB, Vogl AW (2013) A novel
subcellular machine contributes to basal junction remodeling in
the seminiferous epithelium. Biol Reprod 88:60
Dunleavy JEM, O’Bryan MK, Stanton PG, O’Donnell L (2019) The
cytoskeleton in spermatogenesis. Reproduction 157:R53–R72
Egea G, Serra-Peinado C, Salcedo-Sicilia L, Gutiérrez-Martínez
E (2013) Actin acting at the Golgi. Histochem Cell Biol
140:347–360
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

337Histochemistry and Cell Biology (2021) 155:323–340
1 3
Egea G, Serra-Peinado C, Gavilan MP, Rios RM (2015) Cytoskeleton
and Golgi-apparatus interactions: a two-way road of function and
structure. Cell Health Cytoskeleton 7:37–54
Fili N, Hari-Gupta Y, Dos Santos A, Cook A, Poland S, Ameer-Beg
SM, Parsons M, Toseland CP (2017) NDP52 activates nuclear
myosin VI to enhance RNA polymerase II transcription. Nat
Commun 8:1871
Franke WW, Grund C, Fink A, Weber K, Jockusch BM, Zentgraf H,
Osborn M (1978) Location of actin in the microfilament bundles
associated with the junctional specializations between Sertoli
cells and spermatids. Biol Cell 31:7–14
Gotoh N, Yan Q, Du Z, Biemesderfer D, Kashgarian M, Mooseker MS,
Wang T (2010) Altered renal proximal tubular endocytosis and
histology in mice lacking myosin-VI. Cytoskeleton (Hoboken)
67:178–192
Grove BD, Vogl AW (1989) Sertoli cell ectoplasmic specializa-
tions: a type of actin-associated adhesion junctions? J Cell Sci
93:309–323
Grove BD, Pfeiffer DC, Allen S, Vogl AW (1990) Immunofluorescence
localization of vinculin in ectoplasmic (“junctional”) specializa-
tion of rat Sertoli cells. Am J Anat 188:44–56
Guttman JA, Obinata T, Shima J, Griswold M, Vogl AW (2004a) Non-
muscle cofilin is a component of tubulobulbar complexes in the
testis. Biol Reprod 70:805–812
Guttman JA, Takai Y, Vogl AW (2004b) Evidence that tubulobulbar
complexes in the seminiferous epithelium are involved with inter-
nalization of adhesion junctions. Biol Reprod 71:548–559
Haraguchi CM, Mabuchi T, Hirata S, Shoda T, Hoshi K, Yokota S
(2004) Ubiquitin signals in the developing acrosome during sper-
matogenesis of rat testis: an immunoelectron microscopic study.
J Histochem Cytochem 52:1393–1403
Hasson T, Mooseker MS (1994) Porcine myosin-VI: characteriza-
tion of a new mammalian unconventional myosin. J Cell Biol
127:425–440
Hasson T, Walsh J, Cable J, Mooseker MS, Brown SD, Steel KP (1997)
Effects of shaker-1 mutations on myosin-VIIa protein and mRNA
expression. Cell Motil Cytoskeleton 37:127–138
Hayasaka S, Terada Y, Suzuki K, Murakawa H, Tachibana I, Sankai
T, Murakami T, Yaegashi N, Okamura K (2008) Intramanchette
transport during primate spermiogenesis: expression of dynein,
myosin Va, motor recruiter myosin Va, VIIa-Rab27a/b interact-
ing protein, and Rab27b in the manchette during human and
monkey spermiogenesis. Asian J Androl 10:561–568
Hegan PS, Giral H, Levi M, Mooseker MS (2012) Myosin VI is
required for maintenance of brush border structure, composition,
and membrane trafficking functions in the intestinal epithelial
cell. Cytoskeleton 69:235–251
Hegan PS, Lanahan AA, Simons M, Mooseker MS (2015a) Myosin VI
and cardiomyopathy: left ventricular hypertrophy, fibrosis, and
both cardiac and pulmonary vascular endothelial cell defects in
the Snell’s waltzer mouse. Cytoskeleton (Hoboken) 72:373–387
Hegan PS, Kravtsov DV, Caputo C, Egan ME, Ameen NA, Mooseker
MS (2015b) Restoration of cytoskeletal and membrane tethering
defects but not defects in membrane trafficking in the intestinal
brush border of mice lacking both myosin Ia and myosin VI.
Cytoskeleton (Hoboken) 72:455–476
Hicks JL, Deng WM, Rogat AD, Miller KG, Bownes M (1999) Class
VI unconventional myosin is required for spermatogenesis in
Drosophila. Mol Biol Cell 10:4341–4353
Hu J, Cheng S, Wang H, Li X, Liu S, Wu M, Liu Y, Wan X (2019a)
Distinct roles of two myosins in C. elegans spermatid differentia-
tion. PloS Biol 17:e3000211
Hu S, Guo Y, Wang Y, Li Y, Fu T, Zhou Z, Wang Y, Liu J, Pan L
(2019b) Structure of myosin VI/Tom1 complex reveals a cargo
recognition mode of myosin VI for tethering. Nat Commun
10:3459
Inoue T, Kon T, Ohkura R, Yamakawa H, Ohara O, Yokota J, Sutoh K
(2008) BREK/LMTK2 is a myosin VI-binding protein involved
in endosomal membrane trafficking. Genes Cells 13:483–495
Isaji M, Lenartowska M, Noguchi T, Frank DJ, Miller KG (2011) Myo-
sin VI regulates actin structure specialization through conserved
cargo-binding domain sites. PLoS ONE 6:e22755
Kanemori Y, Koga Y, Sudo M, Kang W, Kashiwabara S, Ikawa M,
Hasuwa H, Nagashima K, Ishikawa Y, Ogonuki N, Ogura A,
Baba T (2016) Biogenesis of sperm acrosome is regulated by
pre-mRNA alternative splicing of Acrbp in the mouse. Proc Natl
Acad Sci USA 113:E3696–E3705
Karolczak J, Pavlyk I, Majewski Ł, Sobczak M, Niewiadomski P,
Rzhepetskyy Y, Sikorska A, Nowak N, Pomorski P, Prószyński
T, Ehler E, Rędowicz MJ (2015a) Involvement of unconventional
myosin VI in myoblast function and myotube formation. Histo-
chem Cell Biol 144:21–38
Karolczak J, Sobczak M, Skowronek K, Rędowicz MJ (2015b) A
Kinase anchoring protein 9 Is a novel myosin VI binding partner
that links myosin VI with the PKA pathway in myogenic cells.
Biomed Res Int 2015:816019
Kelleher JF, Mandell MA, Moulder G, Hill KL, L’Hernault SW, Bar-
stead R, Titus MA (2000) Myosin VI is required for asymmetric
segregation of cellular components during C. elegans spermato-
genesis. Curr Biol 10:1489–1496
Kellerman KA, Miller KG (1992) An unconventional myosin
heavy chain gene from Drosophila melanogaster. J Cell Biol
119:823–834
Khawar MB, Gao H, Li W (2019) Mechanism of acrosome biogenesis
in mammals. Front Cell Dev Biol 7:195
Kierszenbaum AL (2002) Intramanchette transport (IMT): managing
the making of the spermatid head, centrosome, and tail. Mol
Reprod Dev 63:1–4
Kierszenbaum AL, Rivkin E, Tres LL (2003a) Acroplaxome, an
F-actin-keratin-containing plate, anchors the acrosome to the
nucleus during shaping of the spermatid head. Mol Biol Cell
14:4628–4640
Kierszenbaum AL, Rivkin E, Tres LL (2003b) The actin-based motor
myosin Va is a component of the acroplaxome, an acrosome-
nuclear-envelope junctional plate, and of manchette-associated
vesicles. Cytogenet Genome Res 103:337–344
Kierszenbaum AL, Tres LL, Rivkin E, Kang-Decker N, van Deursen
JM (2004) The acroplaxome is the docking site of Golgi-
derived myosin Va/Rab27a/b-containing proacrosomal vesicles
in wild-type and Hrb mutant mouse spermatids. Biol Reprod
70:1400–1410
Kierszenbaum AL, Rivkin E, Tres LL (2007) Molecular biology of
sperm head shaping. Soc Reprod Fertil Suppl 65:33–43
Kierszenbaum AL, Rivkin E, Tres LL (2008) Expression of Fer testis
(FerT) tyrosine kinase transcript and distribution sites of FerT
during the development of the acrosome-acroplaxome-manchette
complex in rat spermatids. Dev Dyn 237:3882–3891
Kierszenbaum AL, Rivkin E, Tres LL (2011) Cytoskeletal track selec-
tion during cargo transport in spermatids is relevant to male fer-
tility. Spermatogenesis 1:221–230
Kopera IA, Bilinska B, Chen CY, Mruk DD (2010) Sertoli-germ cell
junctions in the testis: a review of recent data. Philos Trans R Soc
Lond B Biol Sci 365:1593–1605
Kruppa AJ, Kishi-Itakura C, Masters TA, Rorbach JE, Grice GL, Ken-
drick-Jones J, Nathan JA, Minczuk M, Buss F (2018) Myosin
VI-dependent actin cages encapsulate parkin-positive damaged
mitochondria. Dev Cell 44:484–499
Kusumi N, Watanabe M, Yamada H, Li SA, Kashiwakura Y, Matsu-
kawa T, Nagai A, Nasu Y, Kumon H, Takei K (2007) Implica-
tion of amphiphysin 1 and dynamin 2 in tubulobulbar complex
formation and spermatid release. Cell Struct Funct 32:101–113
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

338 Histochemistry and Cell Biology (2021) 155:323–340
1 3
Lenartowska M, Isaji M, Miller KG (2012) A pre-embedding immuno-
gold approach reveals localization of myosin VI at the ultrastruc-
tural level in the actin cones that mediate Drosophila spermatid
individualization. Protoplasma 249:337–346
Li YR, Yang WX (2016) Myosin superfamily: the multi-functional and
irreplaceable factors in spermatogenesis and testicular tumors.
Gene 576:195–207
Li MN, Xiao X, Mruk DD, Lam Y, Lee WM, Lui W, Bonanomi M,
Silvestrini B, Cheng CY (2011) Actin-binding protein drebin E
is involved in junction dynamics during spermatogenesis. Sper-
matogenesis 1:123–136
Li N, Mruk DD, Wong CK, Han D, Lee WM, Cheng CY (2015)
Formin 1 regulates ectoplasmic specialization in the rat testis
through its actin nucleation and bundling activity. Endocrinol-
ogy 156:2969–2983
Lie PPY, Mruk DD, Lee WM, Cheng CY (2009) Epidermal growth
factor receptor pathway substrate 8 (Eps8) is a novel regulator of
cell adhesion and the blood–testis barrier integrity in the semi-
niferous epithelium. FASEB J 23:2555–2567
Lie PPY, Chan AY, Mruk DD, Lee WM, Cheng CY (2010a)
Restricted Arp3 expression in the testis prevents blood-testis
barrier disruption during junction restructuring at spermato-
genesis. Proc Natl Acad Sci USA 107:11411–11416
Lie PPY, Mruk DD, Lee WM, Cheng CY (2010b) Cytoskeletal
dynamics and spermatogenesis. Philos Trans R Soc Lond B
Biol Sci 365:1581–1592
Lin YN, Roy A, Yan W, Burns KH, Matzuk MM (2007) Loss of zona
pellucida binding proteins in the acrosomal matrix disrupts
acrosome biogenesis and sperm morphogenesis. Mol Cell Biol
2:6794–6805
Majewski Ł, Sobczak M, Rędowicz MJ (2010) Myosin VI is asso-
ciated with secretory granules and is present in the nucleus
in adrenal medulla chromaffin cells. Acta Biochim Pol
57:109–114
Majewski Ł, Sobczak M, Wasik A, Skowronek K, Rędowicz MJ (2011)
Myosin VI in PC12 cells plays important roles in cell migration
and proliferation but not in catecholamine secretion. J Muscle
Res Cell Motil 32:291–302
Majewski Ł, Sobczak M, Havrylov S, Jóźwiak J, Rędowicz MJ (2012)
Dock7: a GEF for rho-family GTPases and a novel myosin VI-
binding partner in neuronal PC12 cells. Biochem Cell Biol
90:565–574
Majewski Ł, Nowak J, Sobczak M, Karatsai O, Havrylov S, Lenar-
towski R, Suszek M, Lenartowska M, Redowicz MJ (2018) Myo-
sin VI in the nucleus of neurosecretory PC12 cells: stimulation-
dependent nuclear translocation and interaction with nuclear
proteins. Nucleus 9:125–141
Masters TA, Tumbarello DA, Chibalina MV, Buss F (2017) MYO6
regulates spatial organization of signaling endosomes driving
AKT activation and actin dynamics. Cell Rep 19:2088–2101
Menetrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney
HL, Houdusse A (2005) The structure of the myosin VI motor
reveals the mechanism of directionality reversal. Nature
435:779–785
Mermall V, Miller KG (1995) The 95F unconventional myosin is
required for proper organization of the Drosophila syncytial
blastoderm. J Cell Biol 129:1575–1588
Millo H, Leaper K, Lazou V, Bownes M (2004) Myosin VI plays a role
in cell-cell adhesion during epithelial morphogenesis. Mech Dev
121:1335–1351
Moreno RD, Ramalho-Santos J, Chan EKL, Wessel GM, Schatten G
(2000) The Golgi apparatus segregates from the lysosomal/acro-
somal vesicle during rhesus spermiogenesis: structural alterna-
tions. Dev Biol 219:334–349
Morris SM, Arden SD, Roberts RC, Kendrick-Jones J, Cooper JA,
Luzio JP, Buss F (2002) Myosin VI binds to and localises with
Dab2, potentially linking receptor-mediated endocytosis and the
actin cytoskeleton. Traffic 3:331–341
Morriswood B, Ryzhakov G, Puri C, Arden SD, Roberts R, Dendrou C,
Kendrick-Jones J, Buss F (2007) T6BP and NDP52 are myosin
VI binding partners with potential roles in cytokine signalling
and cell adhesion. J Cell Sci 120:2574–2585
Nakamura N (2013) Ubiquitination regulates the morphogenesis and
function of sperm organelles. Cells 2:732–750
Noguchi T, Miller KG (2003) A role of actin dynamics in individu-
alization during spermatogenesis in Drosophila melanogaster.
Development 130:1805–1816
Noguchi T, Lenartowska M, Miller KG (2006) Myosin VI stabilizes
an actin network during Drosophila spermatid individualization.
Mol Biol Cell 17:2559–2571
Noguchi T, Lenartowska M, Rogat AD, Frank DJ, Miller KG (2008)
Proper cellular reorganization during Drosophila spermatid
individualization depends on actin structures composed of two
domains, bundles and meshwork, that are differentially regulated
and have different functions. Mol Biol Cell 19:2363–2372
O’Donnell L, Nicholls PK, O’Bryan MK, McLachlan RI, Stanton PG
(2011) Spermiation: the process of sperm release. Spermato-
genesis 1:14–35
O’Loughlin T, Masters TA, Buss F (2018) The MYO6 interactome
reveals adaptor complexes coordinating early endosome and
cytoskeletal dynamics. EMBO Rep 19:1–16
Osterweil E, Wells DG, Mooseker MS (2005) A role for myosin VI
in postsynaptic structure and glutamate receptor endocytosis. J
Cell Biol 168:329–338
Pierre V, Martinez G, Coutton C, Delaroche J, Yassine S, Novella
C, Pernet-Gallay K, Hennebicq S, Ray PF, Arnoult C (2012)
Absence of Dpy19l2, a new inner nuclear membrane protein,
causes globozoospermia in mice by preventing the anchoring
of the acrosome to the nucleus. Development 139:2955–2965
Pleuger C, Lehti MS, Dunleavy JE, Fietz D, O’Bryan MK (2020)
Haploid male germ cells—the grand central station of protein
transport. Hum Reprod Update. https ://doi.org/10.1093/humup
d/dmaa0 04
Puri C (2009) Loss of myosin VI no insert isoform (NI) induces a defect
in clathrin-mediated endocytosis and leads to caveolar endocyto-
sis of transferrin receptor. J Biol Chem 284:34998–35014
Puri C, Chibalina MV, Arden SD, Kruppa AJ, Kendrick-Jones J, Buss
F (2010) Overexpression of myosin VI in prostate cancer cells
enhances PSA and VEGF secretion, but has no effect on endo-
cytosis. Oncogene 29:188–200
Qian X, Mruk DD, Cheng YH, Tang EI, Han D, Lee WM, Wong EWP
(2014a) Cheng CY (2014) Actin binding proteins, spermatid
transport and spermiation. Semin Cell Dev Biol 30:75–85
Qian X, Mruk DD, Cheng YH, Tang EI, Han D, Lee WM, Wong EW,
Cheng CY (2014b) Actin binding proteins, spermatid transport
and spermiation. Semin Cell Dev Biol 30:75–85
Raposo G, Marks MS, Cutler DF (2007) Lysosome-related organelles:
driving post-Golgi compartments into specialisation. Curr Opin
Cell Biol 19:394–401
Rikitake Y, Mandai K, Takai Y (2012) The role of nectins in different
types of cell-cell adhesion. J Cell Sci 125:3713–3722
Rivkin E, Kierszenbaum AL, Gil M, Tres LL (2009) Rfn19a, a ubiqu-
tin protein ligase, and Psmc3, a component of the 26S protea-
some, tether to the acrosome membrane and the head-tail cou-
pling apparatus during rat spermatid development. Dev Dyn
238:1851–1861
Rogat AD, Miller KG (2002) A role for myosin VI in actin dynamics
at sites of membrane remodeling during Drosophila spermato-
genesis. J Cell Sci 115:4855–4865
Roux I, Hosie S, Johnson SL, Bahloul A, Cayet N, Nouaille S, Kros CJ,
Petit C, Safieddine S (2009) Myosin VI is required for the proper
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

339Histochemistry and Cell Biology (2021) 155:323–340
1 3
maturation and function of inner ear cell ribbon synapses. Hum
Mol Genet 18:4615–4628
Russell L (1977) Observations on rat Sertoli ectoplasmic (‘junctional’)
specializations in their association with germ cells of the rat tes-
tis. Tissue Cell 9:475–498
Russell L (1979) Further observations on tubulobulbar complexes
formed by late spermatids and Sertoli cells in the rat testis. Anat
Rec 194:213–232
Russell L, Clermont Y (1976) Anchoring device between Sertoli
cells and late spermatids in rat seminiferous tubules. Anat Rec
185:259–278
Sahlender DA, Roberts RC, Arden SD, Spudich G, Taylor MJ, Luzio
JP, Kendrick-Jones J, Buss F (2005) Optineurin links myosin VI
to the Golgi complex and is involved in Golgi organization and
exocytosis. J Cell Biol 169:285–295
Self T, Sobe T, Copeland NG, Jenkins NA, Avraham KB, Steel KP
(1999) Role of myosin VI in the differentiation of cochlear hair
cells. Dev Biol 214:331–341
Shang G, Brautigam CA, Chen R, Lu D, Torres-Vázquez J, Zhang X
(2017) Structure analyses reveal a regulated oligomerization
mechanism of the PlexinD1/GIPC/myosin VI complex. eLife
6:e27322
Siu MK, Wong CH, Xia W, Mruk DD, Lee WM, Cheng CY (2011)
The β1-integrin-p-FAK-p130Cas-DOCK180-RhoA-vinculin is
a novel regulatory protein complex at the apical ectoplasmic
specialization in adult rat testes. Spermatogenesis 1:73–86
Skerget S, Rosenow MA, Petritis K, Karr TL (2015) Sperm proteome
maturation in the mouse epididymis. PLoS ONE 10:e0140650
Sobczak M, Chumak V, Pomorski P, Wojtera E, Majewski Ł, Nowak
J, Yamauchi J, Rędowicz MJ (2016) Interaction of myosin VI
and its binding partner DOCK7 plays an important role in
NGF-stimulated protrusion formation in PC12 cells. Biochim
Biophys Acta 1863:1589–1600
Sriram A, Lyon KRP, Ho CD, Huynh N, Vogl WA (2016) Actin dis-
truptions results in altered morphology of basal tubulobulbar
complexes in rat seminiferous epithelium. Anat Rec (Hoboken)
299:1449–1455
Su W, Mruk DD, Lie PPY, Lui W, Cheng CY (2012) Filamin A is
a regulator of blood-testis barrier assembly during postnatal
development in the rat testis. Endocrinology 153:5023–5035
Sun X, Kovacs T, Hu YJ, Yang WX (2011) The role of actin and
myosin during spermatogenesis. Mol Biol Rep 38:3993–4001
Tardif S, Guyonnet B, Cormier N, Cornwall GA (2012) Alteration
in the processing of the ACRBP/sp32 protein and sperm head/
acrosome malformations in proprotein convertase 4 (PCSK4)
null mice. Mol Hum Reprod 18:298–307
Tokuyasu KT, Peacock WJ, Hardy RW (1972) Dynamic of sper-
miogenesis in Drosophila melanogaster. I Individualization
process. Z Zellforsch Mikrosk Anat 124:479–506
Tomatis VM, Papadopulos A, Malintan NT, Martin S, Wallis T,
Gormal RS, Kendrick-Jones J, Buss F, Meunier FA (2013)
Myosin VI small insert isoform maintains exocytosis by
tethering secretory granules to the cortical actin. J Cell Biol
200:301–320
Tomatis VM, Josh P, Papadopulos A, Gormal RS, Lanoue V, Martin S,
Meunier FA (2017) ENA/VASP proteins regulate exocytosis by
mediating myosin VI-dependent recruitment of secretory gran-
ules to the cortical actin network. Mol Cell Neurosci 84:100–111
Toshimori K (1998) Maturation of mammalian spermatozoa: modifica-
tions of the acrosome and plasma membrane leading to fertiliza-
tion. Cell Tissue Res 293:177–187
Toshimori K (2009) Dynamics of the acrosome. Dynamics of the mam-
malian sperm head: modifications and maturation events from
spermatogenesis to egg activation. Springer, Berlin, Heidelberg,
pp 31–41
Toyama Y (1976) Actin-like filaments in the Sertoli cell junctional spe-
cializations in the swine and mouse testis. Anat Rec 186:477–491
Tumbarello DA, Waxse BJ, Arden SD, Bright NA, Kendrick-Jones J,
Buss F (2012) Autophagy receptors link myosin VI to autophago-
somes to mediate Tom1-dependent autophagosome maturation
and fusion with the lysosome. Nat Cell Biol 14:1024–1035
Tumbarello DA, Kendrick-Jones J, Buss F (2013) Myosin VI and its
cargo adaptors—linking endocytosis and autophagy. J Cell Sci
126:2561–2570
Tumbarello DA, Manna PT, Allen M, Bycroft M, Arden SD, Kendrick-
Jones J, Buss F (2015) The autophagy receptor TAX1BP1 and
the molecular motor myosin VI are required for clearance of Sal-
monella typhimurium by autophagy. PLoS Pathog 11:e1005174
Upadhyay RD, Kumar AV, Ganeshan M, Balasinor NH (2012) Tubu-
lobulbar complex: cytoskeletal remodelling to release sperma-
tozoa. Reprod Biol Endocrinol 10:27
Vaid KS, Guttman JA, Babyak N, Deng W, McNiven MA, Mochizuki
N, Finlay BB, Vogl AW (2007) The role of dynamin 3 in the
testis. J Cell Physiol 210:644–654
Velichkova M, Guttman J, Warren C, Eng L, Kline K, Vogl AW, Has-
son T (2002) A human homologue of Drosophila kelch associ-
ates with myosin-VIIa in specialized adhesion junctions. Cell
Motil Cytoskeleton 51:147–164
Vogl AW, Soucy LJ, Lew GJ (1985) Distribution of actin in isolated
seminiferous epithelia and denuded tubule walls of the rat. Anat
Rec 213:63–71
Vogl AW, Young JS, Du M (2013) New insights into roles of tubulob-
ulbar complexes in sperm release and turnover of blood-testis
barrier. Int Rev Cell Mol Biol 303:319–355
Wang T, Liu NS, Seet LF, Hong W (2010) The emerging role of VHS
domain-containing Tom1, Tom1L1 and Tom1L2 in membrane
trafficking. Traffick 11:1119–1128
Wang H, Wan H, Li X, Liu W, Chen Q, Wang Y, Yang L, Tang H,
Zhang X, Duan E, Zhao X, Gao F, Li W (2014) Atg7 is required
for acrosome biogenesis during spermatogenesis in mice. Cell
Res 24:852–869
Warner CL, Stewart A, Luzio JP, Steel KP, Libby RT, Kendrick-Jones
J, Buss F (2003) Loss of myosin VI reduces secretion and the
size of the Golgi in fibroblasts from Snell’s waltzer mice. EMBO
J 22:569–579
Wells AL, Lin AW, Chen LQ, Safer D, Cain SM, Hasson T, Carragher
BO, Milligan RA, Sweeney HL (1999) Myosin VI is an actin-
based motor that moves backwards. Nature 401:505–508
Wen Q, Wu S, Lee WM, Wong CKC, Lui WY, Silvestrini B, Cheng
CY (2019) Myosin VIIa supports spermatid/organelle transport
and cell adhesion during spermatogenesis in the rat testis. Endo-
crinology 160:484–503
Wollscheid HP, Biancospino M, He F, Magistrati E, Molteni E, Lupia
M, Soffientini P, Rottner K, Cavallaro U, Pozzoli U etal (2016)
Diverse functions of myosin VI elucidated by an isoform-specific
alpha-helix domain. Nat Struct Mol Biol 23:300–308
Wong EWP, Mruk DD, Cheng CY (2007) Biology and regulation of
ectoplasmic specialization, an atypical adherens junction type,
in the testis. Biochim Biophys Acta 1778:692–708
Xiao N, Kam C, Shen C, Jin W, Wang J, Lee KM, Jiang L, Xia J (2009)
PICK1 deficiency causes male infertility in mice by disrupting
acrosome formation. J Clin Invest 119:802–812
Xiao X, Mruk DD, Wong CKC, Cheng CY (2014) Germ cell trans-
port across the seminiferous epithelium during spermatogenesis.
Physiology 29:286–298
Yan HHN, Mruk DD, Lee WM, Cheng CY (2008) Cross-talk between
tight and anchoring junctions—lesson from the testis. Adv Exp
Med Biol 636:234–254
Yang WX, Sperry AO (2003) C-terminal kinesin motor KIFC1 partici-
pates in acrosome biogenesis and vesicle transport. Biol Reprod
69:1719–1729
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

340 Histochemistry and Cell Biology (2021) 155:323–340
1 3
Yang T, Yang WX (2020) The dynamics and regulation of microfila-
ment during spermatogenesis. Gene 744:144635
Yao R, Ito C, Natsume Y, Sugitani Y, Yamanaka H, Kuretake S,
Yanagida K, Sato A, Toshimori K, Noda T (2002) Lack of acro-
some formation in mice lacking a Golgi protein, GOPC. Proc
Natl Acad Sci USA 99:11211–11216
Young JS, Guttman JA, Vaid KS, Shahinian H, Vogl AW (2009) Cort-
actin (CTTN), N-WASP (WASL), and clathrin (CLTC) are pre-
sent at podosome-like tubulobulbar complexes in the rat testis.
Biol Reprod 80:153–161
Young JS, De Asis M, Guttman J, Vogl AW (2012) Cortactin depletion
results in short tubulobulbar complexes and spermiation failure
in rat testes. Biol Open 1:1069–1077
Yu C, Feng W, Wei Z, Miyanoiri Y, Wen W, Zhao Y, Zhang M
(2009) Myosin VI undergoes cargo-mediated dimerization. Cell
138:537–548
Zakrzewski P, Lenartowski R, Rędowicz MJ, Miller KG, Lenartowska
M (2017) Expression and localization of myosin VI in develop-
ing mouse spermatids. Histochem Cell Biol 148:445–462
Zakrzewski P, Rędowicz MJ, Buss F, Lenartowska M (2020a) Loss of
myosin VI expression affects acrosome/acroplaxome complex
morphology during mouse spermiogenesis. Biol Reprod. https
://doi.org/10.1093/biolr e/ioaa0 71
Zakrzewski P, Suwińska A, Lenartowski R, Rędowicz MJ, Buss
F, Lenartowska M (2020b) Myosin VI maintains the actin-
dependent organization of the tubulobulbar complexes required
for endocytosis during mouse spermiogenesis. Biol Reprod
102:863–875
Zhou W, de Iuliis GN, Dun MD, Nixon B (2018) Characteristics of the
epididymal luminal environment responsible for sperm matura-
tion and storage. Front Endocrinol (Lausanne) 9:59
Publisher’s Note Springer Nature remains neutral with regard to
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