Membrane Dynamics during Cytokinesis

Abstract

Endocytic membrane transport has recently emerged as a key process required for the successful completion of cytokinesis. Specific endocytic membranes act in concert with the cytoskeleton and ESCRT proteins to regulate the various stages of cytokinesis. In this review, we focus on the different endocytic Arf and Rab GTPases and their interaction proteins that regulate organelle transport to the intracellular bridge during cytokinesis. The identity and function of these endocytic organelles during the late stages of cell division will also be discussed.

Introduction

The end of the cell cycle culminates in a very complex and highly orchestrated physical separation of daughter cells, known as cytokinesis. It was initially thought that plant and animal cells divide by completely different means. Plant cells divide by building a new cell wall via delivering membranes to a midzone-localized organelle, known as the phragmoblast. In contrast, animal cells were thought to divide solely by using their actomyosin contractile ring, without the need of new membrane delivery. Recent studies clearly demonstrate that membrane transport and subsequent fusion is an important step during cytokinesis in animal cells. Many studies from multiple organisms have shown that membranes originating from the Golgi apparatus and endosomes are trafficked to the intracellular bridge (ICB) of dividing cells and are required for both early and late steps of cytokinesis. The events comprising early cytokinesis have been the subject of many reviews [1–7] and will not be covered at length here. We will focus on membrane transport and its functional significance in regulating late cytokinesis.

Regulation of membrane transport to the intracellular bridge

While membrane transport has been shown to be important in regulating the early phases of cytokinesis, the identity and spatiotemporal properties of identified organelles during late cell division remain unclear [8–11]. Membrane trafficking during cytokinesis was initially investigated in C. elegans and Drosophila embryos by treatment with brefeldin A (BFA) to inhibit anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane (PM) (we will refer to this pathway as the secretory pathway) [12]. It was determined that this secretory pathway is important for cytokinesis, as BFA treatment caused regression of the ICB in C. elegans [13] and Drosophila embryos [14]. Additionally, Gromley et al. [8] and Goss and Toomre [15] both used fluorescently-tagged secretory markers, luminal-GFP and VSVG-YFP respectively, to further establish that secretory vesicles are transported to the ICB during cytokinesis where they fuse with the PM. However, in some experimental models (e.g. the sea urchin embryo), inhibition of the secretory pathway via BFA had no effect on cytokinesis [16]. Furthermore, it was shown that while secretory vesicles are present at the ICB during early telophase, they are largely absent during the abscission step of cytokinesis [11,17]. These results suggest that the secretory pathway may not be the key contributor of membrane during late cytokinesis, and that there must be additional trafficking pathways that are important during cell division.

The importance of recycling endosomes during cytokinesis was originally suggested from work studying the cellularization of fly embryos [18,19]. Subsequently, it was shown that recycling endosomes are important for cytokinesis in mammalian cells [20] and that these endosomes contain Rab11, a small monomeric GTPase required for late cytokinesis [18–20]. In a series of studies, it was discovered that Rab11-binding proteins FIP3 and FIP4 (Rab11 Family of Interacting Proteins 3 and 4) localize to the ICB and simultaneously interact with Rab11 as well as another small GTPase, Arf6 [20–22] (Table 1). Spatiotemporal analysis of FIP3-containing endosomes during cytokinesis demonstrated that FIP3-endosomes are a specific subpopulation of recycling endosomes, are delivered to the ICB during late telophase, and are required for generating a secondary ingression of the ICB to allow for the successful completion of abscission [11,20,23,24]. Finally, it was identified that the FIP3-endosome cargo proteins, SCAMP2/3 and p50RhoGAP, have roles in depolymerizing cortical F-actin in the ICB prior to generation of the secondary ingression [23]. p50RhoGAP presumably achieves decrease in F-actin by inactivating Rho/Rac small GTPases, while the mechanism for SCAMP2/3 remains unclear [23].

Table 1.

Small GTPases and their effector proteins that localize to the intracellular bridge during cytokinesis

Small GTPase	Organelle	Effector Protein (Cytokinesis)	REF
Rab4	Endosome	?	25
Rab8	Secretory/Endosome	?	10, 28
Rab11	Recycling Endosome	FIP3	11, 20–23
		FIP4	20–22
		Sec15 (Exocyst)	8, 21
Rab21	Recycling Endosome	?	27
Rab35	Endosome	OCRL	9, 24
Arf6	Endosome	JIP3	55
		JIP4	55
		FIP3	20–22
		FIP4	20–22
		Sec10 (Exocyst)	8, 21
RalA/RalB	Endosome	Sec5/Exo84 (Exocyst)	56, 57

Echard and colleagues [9] performed siRNA analysis of 29 different Rab GTPases and found that, in addition to Rab11, Rab35 is also required for cytokinesis (Table 1). Rab35 endosomes were shown to be important to maintain the stability of the ICB during cytokinesis and abscission [9] and to influence the localizations of two components known to be required for cytokinesis, phosphatidylinositol-4–5-biphosphate (PtdIns(4,5)P₂) and SEPT2 [9]. Very recently, a PtdIns(4,5)P₂ 5-phosphatase, oculocerebrorenal syndrome of Lowe (OCRL), was identified as a Rab35-binding protein that controls the hydrolysis of PtdIns(4,5)P₂, which provides localized remodeling of the cortical actin network within the ICB [25].

While Rab11 and Rab35 are now well-established regulators that play a key role in cytokinesis, other Rab GTPases such as Rab4, Rab8, Rab14, and Rab21 are also present within either the midzone or the ICB (Table 1) [10,26–28]. Interestingly, none of these GTPases seem to be absolutely required for the completion of cytokinesis, suggesting that they may only play a modulatory role during cell division. Briefly, Rab4, a marker for early endosomes, was found within the central spindle where it co-localizes with Arf6, although the knock-down of Rab4 did not result in a cytokinesis defect [26]. Similarly, Rab8 was shown to localize to the ICB during early cytokinesis [10]. Recently, Dr. Reiner and colleagues [29] demonstrated that Rab8 interacts with cytoplasmic dynein and doublecortin domain-containing protein 5 (DCDC5). DCDC5 is a microtubule-associated protein that, when knocked-down, impairs cytokinesis and Rab8-vesicle positioning in the ICB [29]. However, the depletion of Rab8 does not lead to cytokinesis defects [9] and Rab8-containing organelles are not present within the ICB during abscission [23,30]. Just like Rab4 and Rab8, Rab14 also localizes to endosomes during interphase and is present at the ICB during cytokinesis [27], although the requirement of Rab14 for cell division remains to be demonstrated. Rab21 was recently shown to be involved in the targeted trafficking of integrins into the ICB and that this activity is required for proper cytokinesis, at least in some adherent cell lines [28].

The roles of endosomal membrane transport during cytokinesis

Several studies in a variety of organisms have demonstrated that multiple Rab GTPases define specific subpopulations of endocytic organelles that are differentially transported to the ICB and presumably play distinct roles during cell division. To date, there has not been a comprehensive localization analysis to determine the spatio-temporal dynamics of various endocytic Rabs. As evidenced by recent data (Table 1), specialized populations of organelles are transported to the ICB via different pathways (Figure 1). However, we do not know if and when these endosomes are present at the ICB and the roles that these endosomes play during cytokinesis are only beginning to emerge. The rest of this mini-review will focus on describing the most recent findings about the functions of these different endosomes during cytokinesis.

Figure 1. Endosome trafficking and ESCRT mediated scission of the ICB are required for the completion of cytokinesis.

A model describing the two different mechanisms proposed to regulate the final abscission step in cytokinesis. In late cytokinesis, an actin stabilized intracellular bridge contains three types of endosomes that are known to localize to the intracellular bridge: FIP3-endosomes (Green), Rab35-endosomes (Red) or PI3P-enriched endosomes (Blue). Endosomes mediate ICB actin depolymerization and formation of the secondary ingression to narrow the bridge to ~100 nm. This thinner bridge now is recognized by CHMP4B which marks the abscission site and severs the plasma membrane to complete cytokinesis. The final membrane scission step by occurs through the action of CHMP4B (ESCRT-III).

Membrane fusion is required for cytokinesis

At the beginning of mitosis, during prophase and metaphase, cells round up resulting in the dramatic reduction of PM surface area [31]. This reduction is caused, at least in HeLa cells, by a decrease in recycling endosome fusion with the PM, without any change in the rate of endocytosis [31]. At the completion of anaphase, recycling endosome transport and fusion with the PM resumes, leading to the rapid recovery of daughter cell PM surface area. This regulated change in recycling endosome fusion with the plasma membrane appears to be required for mitosis, since its inhibition leads to cytokinesis defects [31,32]. Additional work has established a similar requirement for endosome fusion with the PM during cytokinesis in multiple other cell lines and organisms [16,33–35]. While it remains incompletely understood why cells need endosomal transport and fusion during cytokinesis, two possible explanations have been put forward regarding the roles of endosomes during division. First, it was suggested that endosomes may provide new membrane and/or specific lipids to the forming ICB. Second, endosomes may allow fast and localized delivery of regulatory proteins leading to the reorganization of the cytoskeleton and PM that is required for abscission to take place. In this mini-review we will not discuss the roles of specific lipids during cytokinesis, since several excellent reviews have been recently published on this topic [36–38]. Instead, we will focus on the endocytic delivery of various regulatory proteins and their roles during abscission.

Endosomes regulate the actin cytoskeleton within the ICB

The actin cytoskeleton plays a key role during the initial formation and ingression of the cleavage furrow, as it acts with non-muscle myosin-II to form an actomyosin contractile ring [2]. Gradual constriction of this actomyosin ring leads to the formation of the ICB. Initially this ICB is stabilized by a cortical actin network, which is derived from the remnants of the actomyosin contractile ring. During late cytokinesis however, this cortical actin cytoskeleton must be disassembled to allow for abscission. One important actin cytoskeleton regulator is PtdIns(4,5)P₂, which is produced de novo inside the ICB via the coordinated action of phosphatase (PTEN) and kinase (PI4K) (see Logan and Mandato [37], and Brill, JA. et al. [36] for further details). Interestingly, OCRL was recently identified as a Rab35-binding protein that is transported to the ICB by Rab35-endosomes [25]. OCRL is a PtdIns(4,5)P₂ 5-phosphatase that was shown to induce actin cytoskeleton disassembly during interphase through the hydrolysis of PtdIns(4,5)P₂ [39]. Consistent with the role of OCRL in abscission, depletion of either Rab35 or OCRL led to increased levels of PtdIns(4,5)P₂ and F-actin within the ICB, as well as abscission defects [25].

Interestingly, Rab35 is not the only endocytic small GTPase that has been shown to regulate F-actin during cytokinesis, as both Rab11 and Arf6 regulate the actin cytoskeleton [19,40,41]. Work from Sullivan’s laboratory has provided evidence that both Rab11 and its effector, Nuf (FIP3 orthologue in fly), are important in modulating actin polymerization in Drosophilia early embryos [19,40]. Mutations in either Rab11 or Nuf inhibit actomyosin contractile ring formation [19,40], suggesting a role for Rab11/Nuf in the initiation of F-actin filament formation/assembly during cellularization in Drosophilia embryos. In contrast, in human cells, Rab11 or FIP3 depletion does not affect actomyosin ring formation or contraction [11,20,21]. Instead, Rab11 and FIP3 function at late cytokinesis, during the abscission of the ICB [11,20,21,24]. It was recently shown that FIP3-endosomes regulate the actin cytoskeleton during the division of mammalian cells by delivering Rho/Rac GTPase activating protein (GAP), p50RhoGAP [23]. When p50RhoGAP is knocked-down in HeLa cells, there is an increase in ICB cortical F-actin causing the delay in abscission [23]. During mammalian cell interphase, Rab11/FIP3 endosomes play a role in cell motility by regulating actin cytoskeleton dynamics within lamellipodia [42]. Furthermore, Arf6, a known FIP3-binding protein, has been revealed to regulate the actin cytoskeleton, at least in part, by activating phosthatidlinositol-4-phosphate-5-kinase to produce PtdIns(4,5)P₂ [43]. Taken together, it is likely that Rab11, Rab35 and Arf6 cooperate to regulate the actin cytoskeleton inside the ICB during the final events leading to abscission.

Endosomes and the ESCRT complexes during the abscission step of cytokinesis

The contraction of the actomyosin ring leads to the formation of the ICB, a structure about 1–3μm in width that connects two daughter cells. The resolution of this ICB is the final abscission step of cytokinesis. Recent high-resolution tomography analyses have demonstrated that the abscission event is preceded by a narrowing of the ICB to ~100nm, a step that is known as the secondary ingression and regulated by FIP3-endosomes [11,23,30,44]. Formation of this secondary ingression is required for abscission, and the mechanisms responsible for this localized ICB thinning are beginning to be revealed. Several years ago it was shown that the ESCRT protein complex is required for abscission, presumably by driving the final scission step in a manner similar to the scission of intraluminal vesicles during multi-vesicular body (MVB) formation, and the budding of enveloped viruses [45–47]. The sequential recruitment of specific ESCRT factors and their proposed function in cytokinesis have been covered in depth elsewhere [44,48]. Briefly, CEP55 is recruited to the midbody early during cytokinesis, shortly after the primary actomyosin-dependent ingression of the plasma membrane, followed by the arrival of Alix and Tsg101 (ESCRT-I) [44]. As the ICB matures, various ESCRT-III components, including CHMP4B, are also recruited to the midbody by binding to the CEP55/Alix/Tsg101 protein complex [44]. During the formation of MVB intraluminal vesicles, polymerization of CHMP4B monomers drives the scission event by constricting the bud neck and bringing its membranes into close proximity for fusion [30,44]. Recently, it was discovered that 17-nm filaments are present within the ICB, and that formation of these filaments is dependent upon CHMP2A (ESCRT-III) [30]. This observation led to the suggestion that the final constriction and abscission of the 1–3μm ICB is mediated by the formation of a continuous ESCRT-III oligomer from the midbody to the abscission site [30]. Recent studies using structured illumination microscopy also demonstrated the sequential association of various ESCRT proteins at the midbody, as well as movement of CHMP4B to the abscission site [44]. Interestingly, a continuous CHMP4B oligomer emanating from the midbody to the abscission site was not observed in these studies [44]. Instead, a disassociated pool of CHMP4B was shown to accumulate at the abscission site [44]. A question that arose from these studies is whether the ESCRT-III complex is actually involved in the formation of the secondary ingression. This question was recently addressed in HeLa cells, where the initiation and formation of the secondary ingression was shown to be dependent upon FIP3-endosome fusion to narrow the ICB to ~100 nm, which is then subsequently stabilized by ESCRT-III to complete abscission (Figure 1). This model is further supported by the fact that the ESCRT-III complex preferentially associates with high curvature membranes (>100 nm) in vitro [49], and is known to sever the narrow 20–50 nm necks of vesicles during MVB biogenesis and viral budding [50–53]. It has been shown that abscission often occurs only on one side of the ICB, thus resulting in one daughter cell inheriting the midbody [8,54]. Since recent studies have indicated that inheritance of the midbody can determine cell fate [54], it would be expected that the establishment of the abscission site is a highly regulated event during mitosis.

Recently it was observed that endosomes also play a role in determining the site of abscission through the initiation of the secondary ingression and possibly by recruiting ESCRT-III (Figure 1) [11,23]. Furthermore, FIP3-dependent delivery of SCAMP2/3 and p50RhoGAP, along with Rab35-dependent delivery of OCRL, are required for the localized disassembly of the actin cytoskeleton within the ICB [25]. It is likely that disassembly of the cortical actin network is required for the formation of the actin-free zone within the ICB, thus leading to increased ICB plasma membrane dynamics (Figure 1). Endosomes potentially can also deliver the components of the ESCRT-III complex to the ICB, as PI3P-containing endosomes transport FYVE-CENT to the ICB of dividing cells [55]. While the function of FYVE-CENT during cell division remains unclear, it was demonstrated that FYVE-CENT forms a complex with TTC19 and CHMP4B [55], thus potentially regulating ESCRT-III assembly at the abscission site (Figure 1). Lastly, recent work has shown that FIP3-endosomes deliver SCAMP3 to the ICB. Since by SCAMP3 interacts with TSG101 [56], FIP3-endosomes may also directly play a role in positioning ESCRT-III at the abscission site. In summary, the delivery and fusion of various endosomes with the ICB plasma membrane are directly involved in disassembly of the actin cytoskeleton to form the secondary ingression and create a region of high membrane curvature to recruit ESCRT-III oligomers for the final scission event (Figure 1).

Conclusions

Current work from many laboratories have demonstrated that abscission is a very complex cellular event that involves coordinated changes in the actin and microtubule cytoskeleton, endocytic membrane transport, ESCRT complex assembly and changes in membrane composition/dynamics. While the requirement for endocytic membranes in cytokinesis has been firmly established, the functions and regulation of these endosomes are only beginning to be uncovered. Thus, the identification and characterization of cargo proteins contained within Rab11/FIP3 and Rab35 endosomes will provide insights into their functional significance. Furthermore, improved high-resolution techniques, combined with time-lapse imaging and appropriate temporal markers will enable the questions presented in this review to be addressed in future studies.