A new role for Rab GTPases during early mitotic stages

Abstract

A recent study revealed new roles for the Rab11 GTPase during mitosis. Rab11 is involved in recycling endosome localization to mitotic spindle poles via dynein-mediated transport. This process is in contrast to Golgi membranes, which disperse in mitosis and do not appear to directly contribute to mitotic functions. Rab11-depletion prevents recycling endosome organization at spindle poles, delays mitotic progression, and disrupts spindle pole protein recruitment, astral microtubule organization, and mitotic spindle orientation. However, Rab11 is not the only endocytic and/or trafficking protein that regulates mitotic progression. Clathrin and two small GTPases (Rab6A’, Rab5) play key roles in spindle organization and function. In this commentary, we discuss the roles of all these canonical endocytic and membrane trafficking proteins during mitosis and speculate on possible cross-communication between them and their molecular pathways that ensure faithful progression through mitosis.

Introduction

It has been known for some time that the recycling endosome is organized around the “centrosome region”¹. However, a direct relationship between these intracellular elements remained unclear until 2012 when electron microscopy revealed an intimate interaction with a substructure of the centrosome, the mother centriole appendages.² Mother centriole appendages have become a focus of interest because they functionally define the “older” centriole of the centriole pair. This has important consequences in biology. For example, the older centriole remains in the stem cell, where Rab11-membranes accumulate, while the cell with the younger centrosome undergoes differentiation.^3,4 The mother centriole also templates cilia formation⁵ and regulates membrane recycling through Rab11.²

The role of the mother centriole was further identified to modulate Rab11-activity through organization of recycling endosome machinery.² If appendages were disrupted, recycling endosome machinery (e.g., the exocyst and the Rab11 GTPase Activating Protein, EVI5) were dispersed.² However, what this study did not demonstrate was whether recycling endosomes contributed to the organization and function of the centrosome. A follow-up study went on to show that the recycling endosome contributes to the organization of the mitotic spindle poles and organization of the spindle.

A Novel Role for Rab11-Endosomes in Spindle Pole Maturation

Earlier work in Caenorhabditis elegans identified a role for Rab11-endosomes during mitosis.^6,7 These studies showed that depletion of Rab11 caused spindle misalignment through disruption of astral microtubule arrays.^6,7 Depletion of RACK1 (receptor of Activated C Kinase 1) or the dynein-interacting protein complex, dynactin, caused the same phenotype. They further demonstrated that RACK1 and dynactin regulated the distribution of recycling endosomes during mitosis^6,7 suggesting a role for Rab11 in regulating spindle alignment and cell cycle progression.

A more recent study showed that Rab11 contributes to spindle pole organization and cell cycle progression in mammalian cells.^8,9 Time-lapse imaging demonstrated that Rab11-endosomes were highly dynamic structures that organized around interphase centrosomes,² mitotic spindles, and mitotic spindle poles.⁹ Rab11 was unexpectedly found in isolated centrosome and spindle pole fractions, making it a bona fide centrosome component. Localization of the GTPase to these mitotic structures required Rab11 to be in its active (Rab11-GTP) form. Even more surprising was the presence of γ-tubulin and dynein⁹ on Rab11-endosomes suggesting that endosomes might act as carriers for microtubule nucleating and spindle pole proteins to mitotic spindle poles. In fact, Rab11-depletion decreased spindle pole-associated γ-tubulin, pericentrin, and endosomes and compromised microtubule regrowth from centrosomes.⁹ Taken together, these studies demonstrated a role for Rab11-endosomes in the maturation of the mitotic spindle pole and thus, the functionality of centrosomes. This study also suggests that endocytic trafficking and perhaps endocytic cargo are modified during mitosis, rather than inactivated, as previously assumed.^10,11 In this regard, Rab11 may be an unexpected new regulator of mitotic progression.

Clathrin Regulates Endocytosis throughout the Cell Cycle and Spindle Maturation during Mitosis

Clathrin was originally identified as an endocytic coat protein that regulated receptor-mediated endocytosis during the growth phase of the cell cycle.¹² It was further proposed that clathrin-mediated endocytosis was disabled during mitotic entry and progression,^10,11 leading to the assumption that the endocytic pathway was inactive during mitosis. However, live cell imaging suggested that endocytosis was not inactive during mitosis and that the rate of endocytosis in mitosis was similar to interphase, although the cell surface area was decreased.¹¹

Other studies demonstrated unique roles for clathrin during mitotic progression and cytokinesis, which were separate from its role in endocytosis.¹³ For example, in mitotic cells, clathrin forms a protein complex with two spindle pole proteins, transforming acidic coiled-coil protein 3 (TACC3) and colonic hepatic tumor overexpressed gene (ch-TOG). These proteins help stabilize the mitotic spindle microtubules,^14-18 an event requiring an interaction with clathrin and clathrin localization to the spindle.^13,15,18

Beyond regulating cell surface area, the reason for continued endocytosis and decreased membrane recycling during mitosis remains elusive. One possibility is that endosomal recycling back to the plasma membrane might be decreased early in mitosis then reactivated upon mitotic exit (anaphase). Another possibility is that microtubule minus-end directed transport to maturing spindle poles is upregulated during early mitosis, to ensure proper spindle pole formation. Finally, we hypothesize that membrane recycling is turned off until late metaphase and/or early anaphase, until the decision is made to commit recycling endosomes to the task of assembling cortical polarity¹⁹ to establish proper spindle orientation and polarity formation in the future daughter cells.^{20, 21}

One problem with studying the role of any regulator of membrane trafficking during the cell cycle is the potential latent effects of protein-loss in earlier cell cycle stages. To combat this problem clathrin was inactivated acutely at specific cell cycle stages by chemical crosslinking of a SNAP-tagged clathrin light chain.¹⁸ Upon crosslinking, clathrin- and ch-TOG recruitment to the mitotic spindle pole was inhibited preventing spindle pole maturation and causing centrosome fragmentation. This suggested a new and important role for clathrin in maintaining centrosome/spindle pole integrity. The authors went on to demonstrate that spindle pole maturation was dependent on a complex of proteins that included clathrin, ch-TOG, and TACC3; and further confirmed the role of Aurora A in formation of this complex.^{16, 18}

In summary, the interaction of clathrin with ch-TOG and TACC3 is essential for centrosome maturation. However, it is still uncertain if the role of clathrin in mitosis and its interaction with ch-TOG are independent of its role as an endocytic protein. Work from Hehnly and Doxsey showed that endosomes are organized at mitotic spindle poles (Fig. 1)⁹ suggesting that endosomes contribute to mitotic functions perhaps through clathrin.^13,18 This could be addressed by testing if: (1) endosomes contribute to clathrin organization at mitotic spindle poles, (2) the interactions between clathrin, ch-TOG, and TACC3 are dependent on Rab11-mediated endosomes, and (3) endosomes and clathrin act independently during mitosis.

Figure 1. Comparison of early-, recycling-endosome, and secretory components, Rab11, Rab5, and Rab6A’, during interphase (A) and metaphase (B).

Rab5 Contributes to Spindle Pole Maturation and Kinetochore Function

Like Rab11, the Rab5 GTPase is involved in the endocytic pathway (Fig. 1) and plays a role in mitotic progression through the regulation of the spindle maturation protein, NuMA,²² and the kinetochore protein, CENP-F(centrosome associated protein).²³ Strikingly similar to disruption of Rab11,⁹ Rab5-depletion caused numerous mitotic-defects such as chromosome misalignment, delays in metaphase and anaphase progression,²² and defects in cytokinesis that resulted in multinucleated cells.^22,23 Again similar to Rab11,⁹ Rab5 localized to endosomes around spindle poles²² (Fig. 1), as demonstrated by the colocalization of GFP-Rab5 with FM4–64-stained membranes and the global endosomal marker, GFP::2xFYVE. In Drosophila, proteomic analysis of Rab5 interacting partners identified MUD (mushroom body defect), the Drosophila homolog of NuMA, further supporting the role of Rab5 in spindle maturation as seen for Rab11.^9,22,23 These observations, suggest unique liaisons between early endosomes (Rab5-labeled) and recycling endosomes (Rab11-labeled) during mitosis. For example, in interphase cells Rab5 endosomes can mature into Rab11 endosomes.²⁴ This membrane maturation process may also occur during mitosis. It is also possible that endosomes involved in mitosis do not mature but remain as distinct entities with distinct tasks.

In addition to spindle pole maturation, Rab5 was shown to play a role in CENP-F localization to kinetochores through the regulation of its release from the nuclear envelope.²³ Mitotic phenotypes associated with Rab5-depletion (e.g., instability of kinetochore fibers, chromosome misalignment, etc.) were similar to CENP-F depletion, suggesting a tight relationship that requires further elucidation. It will be interesting to determine if this relationship is dependent on the spindle pole function of Rab5 or a new and distinct function for the GTPase at kinetochores.

A Non-Membrane Associated, but GTP-Bound, Rab6A´ Regulates Mitotic Progression

Rab6A’ is normally associated with the trans-Golgi apparatus during interphase (Fig. 1),²⁵ but has also been linked to regulation of early mitotic progression.^26,27 Rab6A’ was originally characterized as a regulator of retrograde cargo transport from an endosomal compartment through the Golgi apparatus and on to the endoplasmic reticulum.²⁵ Since the Golgi apparatus vesiculates during mitosis²⁸ it was unclear how Rab6A’ could play a critical role in mitotic progression, although its mitotic function may not require association with the Golgi apparatus. Nevertheless, Rab6A’-depletion caused a decrease in cell number and an increase in the mitotic index due to a metaphase arrest^{26, 27}_. Rab6A’ interacted with several proteins that could regulate mitotic progression. These included GAP-CenA, Rabkinesin-6, and the dynactin subunit, p150^{glued 26, 27}. GAPCenA is a centrosome protein, which contains a Tre-2, Bub2, and Cdc16 (TBC) domain, allowing it to act as a Rab6A’ GAP.²⁷ The kinesin-9 family motor protein, Rabkinesin-6 (a.k.a MKLP2) binds Rab6A’. Rabkinesin-6 is upregulated during mitosis and localizes to the cytokinetic bridge. This motor protein is involved in the assembly of Polo-like-Kinase 1 (Plk1), Cdc14A, and Aurora-B at the mitotic spindle and cytokinetic bridge.^29,30 The dynactin subunit, p150^glued, regulates dynein processivity and is required for facilitating transport of microtubule-nucleating material to the mitotic spindle pole.^31,32 A complex of dynein, dynactin, and Rab6 contributes to the transport of checkpoint proteins, such as Mad2, from kinetochores during mitosis.²² The cross-talk and/or co-functioning between these three established complexes Rab6A’/Rabkinesin-6, Rab6A’/p150^glued, and Rab6A’/GAPCenA, has not been examined. It is interesting to speculate that GAPCenA regulates Rab6A’ activity at the centrosome to either shut down the minus-end microtubule based transport of the Rab6A’/p150 glued complex or to regulate the plus-end mediated transport by Rab6A’/Rabkinesin-6.

The GTP-binding state of Rab6A’ was required for mitotic progression such that cells expressing inactive Rab6A’-GDP arrested in metaphase. However, unlike most active Rab GTPases, Rab6A’-GTP did not seem to associate with a membrane fraction during mitosis, suggesting that Rab6A’ signaling occurs from a cytosolic fraction to promote mitotic progression.²⁶ This raises many intriguing questions. First, if Rab6A’-GTP is indeed non-membrane bound, what are the molecular components that prevent Rab6A’-GTP membrane binding during mitosis? Second, is the prevention of membrane binding required for mitotic progression?

The Endocytic Pathway’s Role in Spindle Orientation and Polarity Establishment

The endocytic system may act as a means to establish proper polarity formation during the cell cycle to ensure appropriate spindle orientation. Spindle orientation is important for organogenesis and development, and when misregulated can contribute to disease pathogenesis (e.g., microcephaly and polycystic kidney disease).³³ For example, in a dividing stem cell within the subventricular zone of the brain, the mother cell stays in the stem cell niche while the daughter cell orients away from the niche where it will ultimately differentiate. Interestingly, Rab11-depleted cells revealed several defects in spindle orientation and the plane of cell division.^7,9 This was caused by a decrease in astral microtubules, a phenotype known to perturb appropriate microtubule contacts with the cell cortex that are required for retention of spindle symmetry. Another possibility that has not yet been tested is whether the perturbation of membrane trafficking by Rab11-depletion causes abnormalities in microtubule attachments at the cortex due to mislocalization of cortical polarity proteins. It will be important to further test the interface between membrane trafficking and cytoskeletal dynamics during mitosis, because the consequence of aberrant divisions is the generation of more differentiated cells at the expense of self-renewing divisions and hence fewer cell numbers.³⁴

Based on these studies, we contend that there is an underlying molecular mechanism for endocytic proteins in mitosis. More detailed studies comparing the role of early endocytic endosomes and recycling endosomes vs. secretory vesicles and their interdependence on one another are required. Recent work along these lines identified over a dozen Rabs involved in mitotic progression. These included Rab37, Rab35, Rab8a, Rab24, Rab7, Rab22a, Rab25, Rab5, Rab6, and Rab11.^35-37 This list adds to the well-studied Rabs (Rab5, Rab6A’, and Rab11) and suggests that these Rab proteins will yield new and important functions for membrane trafficking as it relates to mitotic progression. The list of other endocytic effectors that function in mitosis, such as clathrin, will likely increase as well. This suggests a major role for the endocytic pathway in mitotic function.

10.4161/sgtp.29565

Hehnly H, Doxsey S. Rab11 endosomes contribute to mitotic spindle organization and orientation. Dev Cell. 2014;28:497–507. doi: 10.1016/j.devcel.2014.01.014.