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\DOI{10.5802/crbiol.161}
\datereceived{2024-05-28}
\daterevised{2024-07-16}
\dateaccepted{2024-08-26}
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\dateposted{2024-09-30}
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\CDRsetmeta{articletype}{review}

\title{Incomplete divisions between sister germline cells require Usp8
function}

\alttitle{Les divisions incompl\`{e}tes entre cellules germinales s\oe{}urs requi\`{e}rent Usp8}

\author{\firstname{Juliette} \lastname{Mathieu}\CDRorcid{0000-0002-7152-4245}\IsCorresp}
\address{Coll\`{e}ge de France, PSL Research University, CNRS Biologie,
INSERM, Center for Interdisciplinary Research in Biology, Paris 75005,
France}
\email[J. Mathieu]{juliette.mathieu@college-de-france.fr}

\author{\firstname{Jean-Ren\'{e}} \lastname{Huynh}\CDRorcid{0000-0002-4078-399X}}
\addressSameAs{1}{Coll\`{e}ge de France, PSL Research University, CNRS
Biologie, INSERM, Center for Interdisciplinary Research in Biology,
Paris 75005, France}

\keywords{\kwd{Cytokinesis}\kwd{CHMP4}\kwd{Shrub}\kwd{Deubiquitinating enzyme}}

\altkeywords{\kwd{Cytocin\`{e}se}\kwd{CHMP4}\kwd{Shrub}\kwd{Enzyme de d\'{e}ubiquitination}}

\begin{abstract} 
Cytokinetic abscission is the final step of cell division, resulting in
two separate daughter cells. While abscission is typically complete
across most cell types, germline cells, which produce sexual gametes,
do not finish cytokinesis, maintaining connections between sister
cells. These connections are essential for sharing cytoplasm as they
differentiate into oocyte and sperm. First, we outline the molecular
events of cytokinesis during both complete and delayed abscission,
highlighting the role of the ESCRT-III proteins. We then focus on
recent discoveries that reveal the molecular mechanisms blocking
abscission in \textit{Drosophila} germline cells. The enzyme Usp8 was
identified as vital for ensuring incomplete cytokinesis through the
regulation of ESCRT-III ubiquitination and localization. Finally, we
explore how the processes of incomplete cytokinesis could hold
evolutionary importance, suggesting additional studies into
choanoflagellates to comprehend the origins of multicellularity.
\end{abstract}

\begin{altabstract}
L'abscission est l'\'{e}tape de la cytocin\`{e}se permettant la
s\'{e}paration physique des deux cellules filles \`{a} la fin de la
division cellulaire. Bien que l'abscission soit g\'{e}n\'{e}ralement
compl\`{e}te dans la plupart des types cellulaires, les cellules de la
lign\'{e}e germinale, qui produisent les gam\`{e}tes sexuels, ne
terminent pas leur cytokin\`{e}se, et maintenant des connexions entre
les cellules s\oe{}urs. Ces connexions sont essentielles pour le
partage du cytoplasme requis pour la diff\'{e}renciation en ovocyte ou
en spermatozo\"{i}de. Ici, nous d\'{e}crivons dans un premier temps les
\'{e}v\'{e}nements mol\'{e}culaires de la cytokin\`{e}se lors de
l'abscission compl\`{e}te et retard\'{e}e, en soulignant le r\^{o}le
des prot\'{e}ines ESCRT-III. Nous nous concentrons ensuite sur les
d\'{e}couvertes r\'{e}centes qui r\'{e}v\`{e}lent un m\'{e}canisme
mol\'{e}culaire bloquant l'abscission dans les cellules germinales de
Drosophila. L'enzyme Usp8 est essentielle pour garantir une
cytokin\`{e}se incompl\`{e}te, en r\'{e}gulant les niveaux
d'ubiquitination et la localisation des prot\'{e}ines ESCRT-III. Enfin,
nous explorons comment les processus de cytokin\`{e}se incompl\`{e}te
pourraient avoir une importance \'{e}volutive, sugg\'{e}rant des
\'{e}tudes suppl\'{e}mentaires sur les choanoflagell\'{e}s pour
comprendre les origines de la multicellularit\'{e}.
\end{altabstract}

\def\thanksname{Note}
\thanks{Juliette Mathieu received the Grandes Avanc\'{e}es en Biologie
2023 award}

\maketitle

{\vspace*{9.5pt}}

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\section{Introduction}\label{sec1}

A universal characteristic among all living organisms, from unicellular
to multicellular systems, is the ability to undergo cell division, a
critical process that ensures survival and propagation. Cell division
starts by the duplication of the genetic material and organelles,
followed by their segregation in two daughter cells during cytokinesis.

Cytokinesis is the process that takes place at the end of cell division
and that starts by the invagination of cell membrane between the two
daughter cells. This invagination is driven by the constriction of an
actomyosin ring at the cell cortex. Simultaneously, the mitotic spindle
is remodeled into an electron-dense structure called the midbody.
Abscission then takes place on one side of the midbody with the
cleavage of the membrane and the physical separation of the two
daughter cells~\cite{1,2}. Most eukaryotic cells complete abscission.
In contrast, the development of specific cell types relies on
\mbox{incomplete} cytokinesis, where cells remain connected by stable
intercellular bridges called ring canals to form cysts. The maintenance
of cell connectivity is an evolutionary conserved feature of germ cells
and was also described in some somatic tissues~\cite{3,4,5,6,7,8,9}. In
the male germline, the progression of sperm development leads to the
formation of haploid cells in which the X and Y sexual chromosomes are
segregated in different cells. However, all cells require gene products
from both X and Y chromosomes for their correct differentiation. In
this context, arrested cytokinesis allows X and Y chromosome gene
products to be shared between all cells, although they are genetically
different. Therefore, incomplete cytokinesis in the male germline
complements the genetic content of the future gametes. Most female
germlines also develop as cysts. In meroistic ovaries, only a subset of
germ cells acquire an oocyte fate, while their siblings become nurse
cells. In such cysts, nurse cells transfer material to the future
oocyte through the ring canals to allow its massive growth and
\mbox{differentiation.} Ultimately, nurse cells are eliminated by apoptosis.
This nursing mechanism has been extensively documented in
\textit{Drosophila} female germline, and was more recently described in
mouse ovaries as well. This nursing mechanism is essential in both
species for proper oocyte development as the oocyte is mostly
transcriptionally silent~\cite{10,3}. In mice and fly, mutations
affecting germline cyst formation lead to animal sterility,
highlighting the importance of developmentally regulated incomplete
abscission~\cite{11,12}.

Here, we will briefly review the molecular mechanisms of complete
abscission described in most animal cells and how these molecular
mechanisms are modulated to delay abscission. We will then focus on
abscission in \textit{Drosophila} germline cells, and the recent
discovery of a key enzyme, Usp8, which is both necessary and sufficient
to delay abscission in \textit{Drosophila} germline cells. The results
about Usp8 presented here were originally published in
\textit{Science}~\cite{12} and selected by the Acad\'{e}mie des
sciences. 

\section{Molecular mechanisms of abscission}\label{sec2}

ESCRT-III (for Endosomal Sorting Complex Required for Transport-III)
are small proteins, able to polymerize into helical filaments thought
to be the force driving membrane scission. The ESCRT machinery was
initially identified in
\textit{\href{https://en.wikipedia.org/wiki/Saccharomyces\_cerevisiae}{Saccharomyces 
cerevisiae}} as a group of proteins working sequentially  to
sort membrane receptors into the vacuole~\cite{13,14,15}.  Three
complexes, ESCRT-I, II and III allow the transfer of ubiquitinated
receptors into the lumen of the endosomes through the formation of the
intra-luminal vesicles~\cite{16,17}. In parallel to ESCRT-I and II,
Alix protein also recruits ESCRT-III on endosomes. ESCRT-III proteins
cycle between an inactive closed cytoplasmic state to an open active
conformation, able to polymerize and interact with MIT-domain
containing proteins. Vps4 is an MIT-domain AAA-ATPase that promotes the
dynamic turnover of ESCRT-III filaments required for membrane
scission~\cite{18,19,20,21}. The ESCRT machinery described for the
biogenesis of MVB (multi vesicular bodies)
in yeast is conserved in animal cells, where it is
used in many additional membrane-remodeling processes such as virus
budding, neuron pruning, nuclear envelope reformation, exosomes
release, or cytokinetic abscission. In human cells, CEP55 is localized
at the midbody and recruits Alix and Tsg101, both of which then
mediates the recruitment of ESCRT-III. A secondary pool of ESCRT-III
appears on the side of the midbody, at the site where membrane scission
takes place. Depletion of individual mammalian ESCRT-III proteins, or
of the proteins in charge of their localization delays abscission in
animal  cells~\cite{22,23,24,25,26,27,28}. During abscission, ESCRT-III
turnover in the filaments is regulated by Vps4~\cite{29}. Each
ESCRT-III has the intrinsic ability to form bent filaments, with a
specific curvature, which allows them to form either flat spirals or
helices~\cite{30}. Each ESCRT-III proteins has a selective affinity for
specific membrane curvature and is sequentially recruited on membranes
to promote membrane deformation followed by scission 
{in vitro}~\cite{31,32}. However, how ESCRT-III proteins are organized in
vivo within the cytokinetic bridge is still not fully
understood~\cite{33}. 

In mammalian cells, abscission takes place in G1 phase of the next cell
cycle before the start of DNA replication. However, the timing and
duration of abscission can be coordinated with other cellular events.
For example, in case of spindle defects or lagging chromosomes,
abscission is delayed by Aurora B-dependent checkpoint~\cite{34}. In
addition, this checkpoint is also activated when nuclear pores are not
properly formed or when strong forces are applied on the
bridge~\cite{35,36,37,38}. Several targets of Aurora B have been
described in this process, such as the ESCRT-III
protein~CHMP4C~\cite{39}, as well as \mbox{ANCHR~\cite{40}} or the UKL3
kinase~\cite{41}. All of them localize to the midbody and have the
ability to delay abscission. In the cytoplasm, abscission checkpoint
bodies containing Aurora B, CHMP4C and Alix also delay abscission by
preventing ESCRT-III recruitment at the midbody~\cite{42}.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}(A) Top: female ovariole. Bottom: cell lineage of
the germline stem cell (GSC). (A$'$) Mitotic region of the germarium. At
the end of mitosis, GSCs are connected to their daughter CB through a
ring canal (RC) (GSC \#1) and later by a midbody (MB) (GSC \#2). After
complete cytokinesis, GSCs and CBs are fully separated (GSC \#3). The
fusome (red) links the GSC and the CB before abscission, and also
passes through all RCs of germline cysts. Cap cells (light grey);
terminal filament cells (dark grey). (B)~Cell lineage of a GSC with
delayed abscission. (B$'$) Mitotic region of a germarium when the GSC
exhibit delayed abscission: stem-cysts are detected by the fusome
passing from an anterior GSC to several cells. (C) Cell lineage of a
cyst with ectopic abscission at the 2-cell stage. (C$'$) Mitotic region
of a germarium when a 2-cell cyst exhibit ectopic abscission: a MB is
detected between the two sister cells. (A,A$'$) were also presented
in~\cite{12}; (B,B$'$), (C,C$'$) are original schemes.}
\end{figure*}

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig02}
{\vspace*{3pt}}
\caption{\label{fig2}Confocal images of fixed germaria of control
female (A), female with Usp8 loss of function (LOF) (B,B$'$), female
overexpressing Usp8 (Gain of function, GOF) (C), females with cysts
unable to differenciate (\textit{bam} mutant, (D)), and females with
cysts unable to differenciate but overexpressing Usp8 (E). (B$'$) is a
close-up images of the cysts framed in (B). Dotted lines surround a
GSC/CB pair (A), a stem cyst (C) and a branched fusome indicating
incomplete abscission (E). The white arrow indicates the ectopic
midbody (MB) of a breaking cyst showing complete abcission (B$'$). Scale
bar: 10~${\upmu}$m. (A--C) were also presented in~\cite{12}, (D,E) are
original results.}
{\vspace*{3pt}}
\end{figure*}

\begin{figure*}
\includegraphics{fig03}
{\vspace*{-4pt}}
\bcaption{\label{fig3}(A,B) Confocal images of germaria expressing
GFP-tagged Usp8 from the endogenous locus. (A$'$) and (B$'$) are close-up
of the cells framed in (A) and (B). Dotted lines surround a GSC and its
daughter CB (A$'$), and a germline cyst (B$'$). The white arrow indicates
the MB between the GSC and the CB in (A$'$), and the red arrow indicates
the central ring canal in (B$'$). (C) Immunoblots of Input (In) or
Immunoprecipitated (IP) ubiquitinated proteins from S2 cells
transfected with ESCRT-HA, His-tagged Ubiquitin, and V5-tagged Usp8.
Membranes were blotted with ${\alpha}$-HA antibody, to reveal
ubiquitinated ESCRT-III.}{2}{ Usp8 transfection is sufficient to abolish
CHMP2B and Shrub ubiquitination (red frame). (D,E) Confocal images of
fixed germaria of females expressing Shrub-HA. A GSC/CB pair (D) and a
4-cell cyst (4CC, (E)) are surrounded by dotted lines. The white arrow
in (D) indicates the MB between the GSC and the CB. Shrub-HA is not
detected at the 4CC RC. Scale bar: 5~${\upmu}$m. (F) Confocal images of
a fixed germarium of a female expressing Shrub-HA and lacking Usp8
(Loss of function, LOF). The white arrow indicates the ectopic MB of
the breaking 4CC, the red arrows indicate ectopic Shrub-HA at the RCs
of the mutant 4CC. Scale bar: 5~${\upmu}$m. (G,H) Confocal images of a
fixed ovariole from a female lacking Usp8 (Usp8 LOF) in an otherwise
wild-type (G) or shrb heterozygous background (H). The number of nuclei
of some egg chambers are indicated. Scale bar: 20~${\upmu}$m. (I--K)
Confocal images of fixed germaria with \textit{shrb}$^{-/-}$ germline
cells (GFP negative) expressing Shrb-WT-HA (J), Shrb-KR-HA (K) or no
transgene (I). Dotted lines surround a GSC/CB pair (J) or stem-cysts
(I,K). White arrow indicates the MB decorated by Shrb-WT-HA.
Shrb-KR-HA does not localize at the RC and is unable to rescue the
stem-cyst. Scale bar: 10~${\upmu}$m. These results were originally
presented in~\cite{12}.} 
{\vspace*{2pt}}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}(A) Schematic model of ESCRT levels over time in
the germarium. The GSC has a 24~h cell cycle. Abscission between
the newly formed GSC and its daughter CB occurs late in the cell cycle,
before the next mitosis, and so that the GSC/CB pair never experiences
a Usp8~peak. During this long period, the GSC/CB RC accumulates ESCRT,
reaching a high level allowing abscission. The CB becomes a 16-cell
cyst in 24~h, therefore each cycle lasts only 6~h, leaving much less
time to accumulate ESCRT at RC at each cycle. In addition, Usp8~levels
at the RC in the cyst peak at each mitosis (grey bars) and remove (or
block the dynamic recruitement of) ESCRTs at the RCs. We propose that
short cell cycle duration combined with Usp8 peaks in mitosis are
responsible for the low levels of ESCRT at cysts RC, and absence of
abscission. When Usp8 is absent (red), ESCRT~levels increase at each
cyst cycle, allowing abscission. (B,C) Confocal images and schemes of
fusomes (red) in germaria of females expressing CHMP2B-GFP in a WT
context (B) or when cysts exhibit a prolonged interphase, from T0, to
10 and 20~h (C). In (C), 4-cell cysts are observed at T0 (left).
CHMP2B-GFP is then observed at the RCs (red arrow) of a 4-cell cyst
(${+}$10~h, middle), and the central RC of a 4-cell cyst breaks into a
MB (white arrow) at T0 ${+}$20~h (right). Scale~bar: 5~${\upmu}$m.
These results were originally described in~\cite{12}.}
{\vspace*{2pt}}
\end{figure*}

\section{Abscission in \textit{Drosophila} germline cells} \label{sec3}

Apart from being induced by cellular defects, delayed cytokinesis is
also observed in many developmental contexts, and is a conserved feature
of germline cells~\cite{9}. Because of the powerful genetics of
\textit{Drosophila} and its well-described \mbox{oogenesis,}
\textit{Drosophila} germline is an established model to {understand}
incomplete cytokinesis. In adult females, germline stem cells (GSC)
divide every day at the tip of a specialized structure called the
germarium. These divisions are complete, even though abscission in
these cells takes place late, in G2 of the next cell
cycle~\cite{27,43,44}. The division of the GSC is asymmetric and
generates a new GSC and a cystoblast (CB). The differentiation program
of the cystoblast involves four rounds of synchronous divisions with
incomplete cytokinesis and leads to the formation of a 16-cell cyst
(Figure~\ref{fig1}A). Once the cyst is formed, one cell is specified as
the oocyte and the 15 others become nurse cells. These cysts then
mature as egg chambers (Figure~\ref{fig1}A). The molecular mechanisms
that regulate abscission in the GSC have started to be elucidated, and
the involvement of the ESCRT machinery was demonstrated. In fly, Alix
is recruited directly by Pavarotti/MKLP1~\cite{45}. Although complete
loss of Alix is viable, females are sterile and their GSCs exhibit a
strong abscission delay~\cite{28}. Shrub is the single and essential
\textit{Drosophila} homolog of CHMP4; removing one copy of the Shrub
gene is sufficient to induce a strong GSC abscission delay~\cite{27}.
As in mammalian cells, Aurora B activity delays abscission in GSCs.
Abscission delay of the GSCs can be visualized by the formation of
``stem cysts'', made of sister cells having a GSC identity (``stem-'')
but which remain connected by ring canals (``-cyst'')~\cite{46}. In
these stem-cysts, the fusome, an ER derived organelle specific to the
germline, passes through the ring canals as in wild-type cysts
(Figure~\ref{fig1}B). Eventually, abscission occurs and releases a
cystoblast made of two cells instead of one cell. Then, this two-cell
cystoblast enters the differentiation program and goes through four
incomplete divisions leading to the formation of 32-cell cysts instead
of 16 (Figure~\ref{fig1}A,B). Conversely, in mutant females for a
hypomorphic allele of Aurora~B (low Aurora B activity), abscission
occurs ectopically in germline cysts instead of remaining
incomplete~\cite{46}. The outcome of these ectopic abscissions is the
formation of germline cysts made of 8 cells or less
(Figure~\ref{fig1}C). 

To gain insights into the molecular mechanisms that regulate the
incomplete abscission of the germline cysts, we performed a genetic
screen. We knocked down potential ESCRT-III interactors in
\textit{Drosophila} germline cells, and analyzed the numbers of cells
in each egg chamber. With this simple assay, we identified the
gene~\textit{Usp8} as necessary for incomplete abscission: cysts
lacking Usp8 undergo complete abscission, and egg chambers of 8 cells
or less are formed as a consequence (Figure~\ref{fig2}A,B). On the
other hand, over-expression of Usp8 is \mbox{sufficient} to delay abscission
in GSCs, as revealed by the appearance of stem-cysts and the formation
of 32-cell egg chambers (Figure~\ref{fig2}C). Over-expression of Usp8
is also sufficient to block abscission in germline cells that are
unable to differentiate into cysts (Figure~\ref{fig2}D,E). Therefore,
we concluded that Usp8 is \mbox{necessary} and sufficient to promote
incomplete cytokinesis in \textit{Drosophila} germline cells.

We next investigated the molecular mechanism underlying the ability of
Usp8 to block cytokinesis in germline cysts. We tagged endogenous Usp8
with GFP and found that it localizes at ring canals in both GSCs and
germline cysts (Figure~\ref{fig3}A,B). Usp8 is a deubiquitinating
enzyme that removes ubiquitin moiety on its substrates. On the one
hand, biochemical experiments showed that Shrub, CHMP2B and CHMP1,
three ESCRT-III proteins, are all ubiquitinated in \textit{Drosophila}
cells~\cite{12}. On the other hand, we showed that Usp8 is able to
deubiquitinate Shrub and CHMP2B (Figure~\ref{fig3}C). In wild type
germline cysts, neither Shrub nor CHMP2B are enriched at the ring
canals linking the sister cells. However, both proteins become enriched
in the absence of Usp8 (Figure~\ref{fig3}D--F and~\cite{12}). These
results indicate that deubiquitination of ESCRT-III by Usp8 is
necessary to block their enrichment at ring canals. To test if this
ectopic ESCRT-III localization is responsible for the complete
abscission observed in Usp8 mutant germline cysts, we reduced Shrub or
CHMP2B levels in Usp8 depleted cysts. In this double-mutant context, we
found less ectopic abscissions in cysts, and egg chambers were formed
with more cells at later stages (Figure~\ref{fig3}G,H). Altogether, we
concluded that Usp8 is required in cysts to \mbox{deubiquitinate} \mbox{ESCRT-III,}
to block their recruitment at the ring canals and subsequent
abscission. This result also raised the question of the importance of
ubiquitination of ESCRT-III during complete abscission. To address this
issue, we analyzed the ability of a non-ubiquitinatable form of Shrub
(Shrub-KR) to rescue the abscission delay {phenotype} induced by a loss
of Shrub in GSCs. We found that unlike wild type Shrub,
non-ubiquitinatable Shrub is unable to localize to the ring canal and
to promote abscission (Figure~\ref{fig3}I--K). This result indicates
that ubiquitination of Shrub is necessary for its proper localization
and function during GSC \mbox{abscission.}

Our data showed that Usp8 blocks the accumulation of ESCRT-III at the
ring canals of germline cysts. However, ESCRT-III proteins do
accumulate at the GSC ring canal despite the presence of Usp8. So, the
presence or absence of Usp8 alone cannot explain the difference between
complete and incomplete cytokinesis. We hypothesized that the
difference of cell cycle duration between GSCs and germline cysts could
explain this apparent contradiction. Indeed, each GSC divides only
every 20~h, its abscission is very long and lasts until the
G2~phase of the next cell cycle. By contrast, the four cycles leading
to the formation of a 16-cell cyst last 24~h, that is, 6~h for
each cycle. We analyzed the levels of the ESCRT-III CHMP2B after
mitosis of the GSCs, and found that it increases progressively until
abscission occurs. By contrast, no accumulation is observed in wild
type germline cysts. In cysts lacking Usp8, CHMP2B levels accumulate
with time (Figure~\ref{fig4}A). In addition, we also noted a burst of
Usp8 levels at cyst ring canals during each mitosis
(Figure~\ref{fig4}A). We therefore speculated that the long cell \mbox{cycle}
of GSCs allows accumulation of ESCRT-III, while the short cell cycle in
the cyst, combined with mitotic peaks of Usp8, prevents ESCRT-III
accumulation and subsequent abscission (Figure~\ref{fig4}A). To test
experimentally this model, we used mutant flies with prolonged
interphases \mbox{specifically} in germline cysts. In this background, cysts
were initially devoid of ESCRT-III as in wild type cells
(Figure~\ref{fig4}B,C). However, when observed 10~h later, most
cysts exhibited CHMP2B enrichment at their ring canals. After 10 more
hours, the levels of CHMP2B increased and cysts were breaking apart.
This result showed that time is a critical parameter for ESCRT-III
localization, allowing their accumulation at ring canals and subsequent
abscission.

Altogether, our results showed that the switch between complete and
incomplete cytokinesis depends on the regulation of ESCRT-III
ubiquitination levels by Usp8, coordinated with cell-specific cell
cycle durations. These results were published in~\cite{12}.

\section{Perspectives}\label{sec4}

This work revealed that ubiquitination of ESCRT-III proteins promotes
the completion of abscission in \textit{Drosophila} GSCs, as it allows
their accumulation at the abscission site. Interestingly,
ubiquitination of ESCRT is also required for complete abscission in
archeal cells, suggesting that this post-translational modification has
a conserved role during evolution~\cite{47}. However, this
ubiquitination leads to ESCRT degradation in archaea, whereas ESCRT are
stabilized by ubiquitination in \textit{Drosophila} germline cells.
This raises the question of whether ESCRT stabilization by
ubiquitination appeared with multicellular organisms. Choanoflagellates
are the closest relatives to animals and have the ability to switch
from unicellular form to multicellular colonies by incomplete
divisions. Exploring the functions of Usp8 and ESCRT ubiquitination in
choanoflagellates could thus reveal exciting insights into the origin
of multicellularity. 

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

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