**4. Unconventional function of membrane trafficking proteins in mitosis**

Recent findings have shown that clathrin-mediated endocytosis is active throughout mitosis, while the recycling pathway slows down from prophase until the completion of anaphase (Boucrot & Kirchhausen, 2007). These data have been generated by monitoring the changes in plasma membrane area during mitosis in living cells with a membrane-impermeant dye that becomes fluorescent upon binding to the outer leaflet of the plasma membrane. Since the dye cannot flip to the inner leaflet, only endocytic vesicles generated by internalization of the plasma membrane can be visualized. At metaphase, these plasma membrane-derived vesicles are not delivered back at the surface resulting in a net decrease of the cell area. In turn, this translates in cell detachment and round up from prophase to anaphase. The recycling pathway recovers at telophase when the forming daughter cells start to spread again (Boucrot & Kirchhausen, 2007).

Interestingly, mitotic phosphorylation of Rab4, a GTPase required for recycling from early endosomes to the plasma membrane (van der Sluijs et al, 1992), prevents its localization at

composition of the machineries that control active *vs.* inactive integrin traffic could imply that higher amounts of endocytic proteins are required to effectively internalize ECM-bound integrins. Accordingly, the force-generating retrograde motor Myo6 (Spudich & Sivaramakrishnan, 2010) participates to endocytosis, transport of endosomal vesicles along F-actin (Hasson, 2003), and active integrin internalization (Valdembri et al, 2009) as well.

Clathrin coats exist either as classical curved clathrin-coated pits or as flat clathrin-coated plaques that depend on the presence of the actin cytoskeleton and occur only at ECMadherent surfaces, indicating that integrin-mediated adhesion of cells to the ECM likely control the organization of the different clathrin-based endocytic structures (Kirchhausen, 2009; Saffarian et al, 2009). The potential role of cell-to-ECM adhesion in regulating clathrinmediated endocytosis is further supported by the recent experimental observation that the closer clathrin-coated pits are to integrin-containing adhesion sites the slower are their internalization dynamics (Batchelder & Yarar, 2010). It is indeed possible that the binding of integrins to the ECM could give rise to forces that counteract the pulling forces required to deform and curve the PM to finally allow clathrin-based internalization. Such a hypothesis could also account for the requirement of different molecular complexes for active *vs.*

To date, only few proteins have been selectively involved in inactive, but not active, integrin traffic and the degree of specificity for the bent/inactive integrin conformation is still matter of debate. A prominent example is represented by the endocytic adaptor protein disabled 2 (DAB2), that is able to directly bind the cytodomain of integrin β subunits (Calderwood et al, 2003), and was recently found to selectively promote the internalization of inactive β1 integrins (Teckchandani et al, 2009). However, during ECM-adhesion disassembly experiments, Chao and Kunz, by incubating living cells with the anti-active β1 integrin monoclonal antibody 12G10, found that active β1 integrins could be endocytosed in a DAB2-dependent manner as well (Chao & Kunz, 2009). However, since incubation of living cells with function activating or blocking antibodies represents a significant bias in the study of integrin activation physiology, further work is needed to better characterize the role of

**4. Unconventional function of membrane trafficking proteins in mitosis** 

Recent findings have shown that clathrin-mediated endocytosis is active throughout mitosis, while the recycling pathway slows down from prophase until the completion of anaphase (Boucrot & Kirchhausen, 2007). These data have been generated by monitoring the changes in plasma membrane area during mitosis in living cells with a membrane-impermeant dye that becomes fluorescent upon binding to the outer leaflet of the plasma membrane. Since the dye cannot flip to the inner leaflet, only endocytic vesicles generated by internalization of the plasma membrane can be visualized. At metaphase, these plasma membrane-derived vesicles are not delivered back at the surface resulting in a net decrease of the cell area. In turn, this translates in cell detachment and round up from prophase to anaphase. The recycling pathway recovers at telophase when the forming daughter cells start to spread

Interestingly, mitotic phosphorylation of Rab4, a GTPase required for recycling from early endosomes to the plasma membrane (van der Sluijs et al, 1992), prevents its localization at

inactive integrin internalization.

again (Boucrot & Kirchhausen, 2007).

DAB2.

endosomal membranes (Ayad et al, 1997). During mitosis, phosphorylated Rab4 is in the cytosol complexed with the peptidyl-prolyl isomerase Pin1 and it is no longer able to recruit downstream effectors on endosomes (Gerez et al, 2000). Thus an appealing possibility is that Rab4 phosphorylation might participates in the inhibition of the recycling pathway measured by Boucrot and Kirchhausen during the early steps of mitosis.

Of note, fusion of early endosomes in mitosis is blocked via cdc2-dependent phosphorylation events (Tuomikoski et al, 1989). This might represent an additional mechanism to inhibit vesicles recycling at the plasma membrane by altering the homeostasis of the endosomal compartment and affecting the generation of exocytic vesicles. Inhibition of homotypic fusion of early endosomes at mitosis is also caused by decreased residence time of the early endosome-tethering molecule EEA1 on endosomal membranes (Bergeland et al, 2008). It would be interesting to define how the acceleration of the EEA1 cycle between cytosol and membranes is achieved in mitotic cells.

Endocytic/trafficking proteins are also emerging as important factors required for the proper execution of cell division. Beside the involvement of trafficking molecules in membrane delivery to the cleavage furrow at cytokinesis [for recent reviews see (McKay & Burgess, 2011; Montagnac et al, 2008)], some of these proteins also display specific functions in mitosis. Here we will review knowledge rising on this issue.

One of the best-characterized endocytic molecules showing a distinct role in mitosis is the clathrin heavy chain. The clathrin complex is organized in a triskelion made of three heavy chains each with an associated light chain (ter Haar et al, 1998). At metaphase, clathrin also localizes to kinetochore fibers (spindle microtubules connecting kinetochores to spindle poles) of the spindle apparatus (Royle et al, 2005). Here it stabilizes spindle microtubules aiding congression of chromosomes on the metaphase plate. Depletion of clathrin heavy chain by RNA interference causes failure in the correct attachment of chromosomes to kinetochore fibers resulting in misaligned chromosomes and in persistent activation of the mitotic checkpoint thus prolonging mitosis (Royle et al, 2005). More recently, some advances in understanding clathrin function at the spindle have been made. Clathrin heavy chain has been found to bind to TACC3, phosphorylated on serine 558 by Aurora A, and to recruit it to the spindle. In turn, TACC3 is responsible for localization of ch-TOG, a protein that promotes microtubule assembly and spindle stability, to spindles (Lin et al, 2010). In agreement, functional ablation of clathrin heavy chain causes loss of ch-TOG from spindles and destabilizes kinetochore fibers affecting chromosome congression. Based on electron microscopy data, it has been proposed that TACC3/ch-TOG/clathrin heavy chain complex works as an inter-microtubules bridge that stabilizes kinetochore fibers by physical crosslinking reducing the rate of microtubule catastrophe (Booth et al, 2011).

Another important endocytic player is epsin, an adaptor molecule that binds and deforms membranes driving curvature of clathrin-coated pits (Ford et al, 2002). At mitosis, epsin participates in spindle morphogenesis indirectly through its ability to regulate mitotic membrane organization (Liu & Zheng, 2009). In cells depleted of epsin, by RNAi-mediated silencing, the membrane network that uniformly surrounds the chromosomes is distorted with uneven membrane distribution frequently showing layers of membrane whorls. This, in turn, alters spindle morphology resulting in splayed spindle poles and multipolar spindles (Liu & Zheng, 2009).

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 171

(Audhya et al, 2007; Serio et al, 2011). Although the molecular mechanisms are unclear, it has been proposed that Rab5 might act *in trans*, while localized on endosomes, by interacting with effectors on the ER membrane to induce their homotypic fusion. Furthermore, recent findings have shown that, at mitosis, Rab5 is required for proper chromosome alignment both in human cells and in the *Drosophila* system (Serio et al, 2011;

One relevant question is whether the modality of function for these molecules at mitosis is distinct from their role in membrane trafficking during interphase. A couple of observations

First, some of these proteins display a subcellular localization in mitosis distinct from trafficking membranes. For instance, the globular N-terminal domain of the clathrin heavy chain is responsible for clathrin localization to kinetochore fibers and a number of assays, including labeling of intracellular membranes, electron microscopy analysis and mass spectrometry, revealed that it does not coat membranes at the spindle but it rather bind to microtubules or to microtubules-associated proteins (Royle et al, 2005). Localization of dynamin to centrosome, which is a non-membranous organelle, is dynamic and occurs through its middle domain in a microtubules-independent manner (Thompson et al, 2004). Second, these molecules appear to interact with binding partners distinct from those involved in vesicular trafficking pathways and such interactions seem to be relevant during cell division. Indeed, clathrin has been reported to bind and stabilize spindle microtubules (Royle et al, 2005) while dynamin interacts with the centrosomal protein γ-tubulin (Thompson et al, 2004). In addition, the β2-adaptin subunit of the clathrin adaptor AP2 associates, at least in vitro, with a component of the mitotic spindle checkpoint, the kinase BubR1. Although the physiological meaning of this interaction is unknown, it might provide a link between endocytic proteins and the mitotic checkpoint machinery (Cayrol et al, 2002). Of note, two accessory components of clathrin coated pits, epsin and Eps15 are phosphorylated at mitosis and such modification reduces their binding to the α-adaptin subunit of AP2 (Chen et al, 1999). Among the different hypothesis that can be envisioned, one appealing possibility is that mitotic phosphorylation of epsin and Eps15 alters their binding capabilities promoting formation of protein complexes working at mitosis and involving partners distinct from AP2. Importantly, epsin has been shown to facilitate spindle organization independently from its endocytic function by using cell-free spindle assembly assays. In these assays, *Xenopus* egg extracts, lacking the membrane cortex, have been depleted of epsin and reconstituted with purified epsin or with epsin lacking the membrane-bending domain. Only full length epsin was able to rescue the spindle defects demonstrating that the membrane curvature activity of epsin is required for the establishment of spindle morphology independently from endocytosis (Liu & Zheng, 2009). This study nicely extends the concept that endocytic proteins have a role in mitosis distinct

Given that endocytosis is active throughout the cell cycle and that, at mitosis, some endocytic proteins are also involved in pathways different from internalization, these molecules might play two distinct functions simultaneously thus coordinating membrane

Capalbo et al, 2011).

argue in favor of this possibility.

from the one exerted during interphase.

traffic with the execution of mitotic events.

Huntingtin-interacting protein 1-related (HIP1r) functions in clathrin-mediated endocytosis and links endocytosis to the actin cytoskeleton (Engqvist-Goldstein et al, 2001). HIP1r also localizes to the spindle and its depletion by RNA interference causes chromosome misalignment and activation of the spindle checkpoint (Park, 2011).

In addition, is worth to mention that Rab6A', a GTPase that regulates trafficking between the Golgi and post-Golgi membrane compartments, is also required for spindle stability (Mallard et al, 2002). At mitosis, depletion of Rab6A' arrests cells at metaphase (Miserey-Lenkei et al, 2006). Aligned chromosomes, in Rab6A'-depleted cells, show increased amount of p150Glued, a subunit of the dynein/dynactin complex, and of Mad2 at kinetochores. p150Glued takes part in the release of the checkpoint protein Mad2 from kinetochores thus switching off the mitotic checkpoint, an operation required for the transition of cells from metaphase to anaphase. The inability of Rab6A'-silenced cells to progress mitosis might be the consequence of defective p150Glued-mediated transport of Mad2 out of kinetochores resulting in the failure to turn off the checkpoint. Thus Rab6A', by regulating the dynamics of the dynein/dynactin complex at the kinetochores, cooperates to the inactivation of the Mad2-spindle checkpoint.

Some trafficking proteins have also been found to act at the centrosome which is part of the mitotic machinery that ensure proper chromosome segregation. One of these proteins is dynamin. In addition to its membrane localization, dynamin is at the centrosome throughout the cell cycle and localizes to the spindle midzone and to the cleavage furrow during cytokinesis (Thompson et al, 2004; Thompson et al, 2002). Depletion of dynamin by RNA interference causes centrosome separation indicating a role for dynamin in the maintenance of centrosome cohesion (Thompson et al, 2004).

The Autosomal Recessive Hypercholesterolemia (ARH) protein provides another example. ARH is a cargo-specific adaptor that functions in clathrin-mediated endocytosis of receptors of the LDLR family (Shin et al, 2001). It displays a complex subcellular localization being on endocytic vesicles and at the centrosome in interphase. During mitosis, it also localizes to kinetochores, spindle poles and midbody. The suggested function for ARH is in centrosome assembly, as ARH-/- embryonic fibroblasts show smaller centrosomes. Since ARH binds to the dynein motor protein it could cooperate in the transport of components to the centrosome. Of note, functional ablation of ARH also strongly delays cytokinesis (Lehtonen et al, 2008).

In addition, the Rab-GAP protein RN-tre is phosphorylated at mitosis and dephosphorylated by the dual-specificity phosphatase Cdc14A (Lanzetti et, 2007). Cdc14A controls key mitotic events and it is also implicated in centrosome function in human cells (Mailand et al, 2002). Mitotic phosphorylation on RN-tre modulates its GAP activity establishing an additional link between endocytosis and the machinery working at mitosis (Lanzetti et al, 2007).

Finally, Rab5 is required for nuclear membrane breakdown at mitosis, as depletion of this GTPase in *C. elegans* delays nuclear envelope disassembly and the release of nuclear envelope and lamina components (Audhya et al, 2007). The activity of Rab5 in nuclear envelope disassembly appears to result from its involvement in structuring the ER, of which the nuclear membrane represents a functional district (Audhya et al, 2007). Rab5 participates to mitotic ER clustering and to disassembly of the nuclear envelope also in mammalian cells

Huntingtin-interacting protein 1-related (HIP1r) functions in clathrin-mediated endocytosis and links endocytosis to the actin cytoskeleton (Engqvist-Goldstein et al, 2001). HIP1r also localizes to the spindle and its depletion by RNA interference causes chromosome

In addition, is worth to mention that Rab6A', a GTPase that regulates trafficking between the Golgi and post-Golgi membrane compartments, is also required for spindle stability (Mallard et al, 2002). At mitosis, depletion of Rab6A' arrests cells at metaphase (Miserey-Lenkei et al, 2006). Aligned chromosomes, in Rab6A'-depleted cells, show increased amount of p150Glued, a subunit of the dynein/dynactin complex, and of Mad2 at kinetochores. p150Glued takes part in the release of the checkpoint protein Mad2 from kinetochores thus switching off the mitotic checkpoint, an operation required for the transition of cells from metaphase to anaphase. The inability of Rab6A'-silenced cells to progress mitosis might be the consequence of defective p150Glued-mediated transport of Mad2 out of kinetochores resulting in the failure to turn off the checkpoint. Thus Rab6A', by regulating the dynamics of the dynein/dynactin complex at the kinetochores, cooperates to the inactivation of the

Some trafficking proteins have also been found to act at the centrosome which is part of the mitotic machinery that ensure proper chromosome segregation. One of these proteins is dynamin. In addition to its membrane localization, dynamin is at the centrosome throughout the cell cycle and localizes to the spindle midzone and to the cleavage furrow during cytokinesis (Thompson et al, 2004; Thompson et al, 2002). Depletion of dynamin by RNA interference causes centrosome separation indicating a role for dynamin in the

The Autosomal Recessive Hypercholesterolemia (ARH) protein provides another example. ARH is a cargo-specific adaptor that functions in clathrin-mediated endocytosis of receptors of the LDLR family (Shin et al, 2001). It displays a complex subcellular localization being on endocytic vesicles and at the centrosome in interphase. During mitosis, it also localizes to kinetochores, spindle poles and midbody. The suggested function for ARH is in centrosome assembly, as ARH-/- embryonic fibroblasts show smaller centrosomes. Since ARH binds to the dynein motor protein it could cooperate in the transport of components to the centrosome. Of note, functional ablation of ARH also strongly delays cytokinesis (Lehtonen

In addition, the Rab-GAP protein RN-tre is phosphorylated at mitosis and dephosphorylated by the dual-specificity phosphatase Cdc14A (Lanzetti et, 2007). Cdc14A controls key mitotic events and it is also implicated in centrosome function in human cells (Mailand et al, 2002). Mitotic phosphorylation on RN-tre modulates its GAP activity establishing an additional link between endocytosis and the machinery working at mitosis

Finally, Rab5 is required for nuclear membrane breakdown at mitosis, as depletion of this GTPase in *C. elegans* delays nuclear envelope disassembly and the release of nuclear envelope and lamina components (Audhya et al, 2007). The activity of Rab5 in nuclear envelope disassembly appears to result from its involvement in structuring the ER, of which the nuclear membrane represents a functional district (Audhya et al, 2007). Rab5 participates to mitotic ER clustering and to disassembly of the nuclear envelope also in mammalian cells

misalignment and activation of the spindle checkpoint (Park, 2011).

maintenance of centrosome cohesion (Thompson et al, 2004).

Mad2-spindle checkpoint.

et al, 2008).

(Lanzetti et al, 2007).

(Audhya et al, 2007; Serio et al, 2011). Although the molecular mechanisms are unclear, it has been proposed that Rab5 might act *in trans*, while localized on endosomes, by interacting with effectors on the ER membrane to induce their homotypic fusion. Furthermore, recent findings have shown that, at mitosis, Rab5 is required for proper chromosome alignment both in human cells and in the *Drosophila* system (Serio et al, 2011; Capalbo et al, 2011).

One relevant question is whether the modality of function for these molecules at mitosis is distinct from their role in membrane trafficking during interphase. A couple of observations argue in favor of this possibility.

First, some of these proteins display a subcellular localization in mitosis distinct from trafficking membranes. For instance, the globular N-terminal domain of the clathrin heavy chain is responsible for clathrin localization to kinetochore fibers and a number of assays, including labeling of intracellular membranes, electron microscopy analysis and mass spectrometry, revealed that it does not coat membranes at the spindle but it rather bind to microtubules or to microtubules-associated proteins (Royle et al, 2005). Localization of dynamin to centrosome, which is a non-membranous organelle, is dynamic and occurs through its middle domain in a microtubules-independent manner (Thompson et al, 2004).

Second, these molecules appear to interact with binding partners distinct from those involved in vesicular trafficking pathways and such interactions seem to be relevant during cell division. Indeed, clathrin has been reported to bind and stabilize spindle microtubules (Royle et al, 2005) while dynamin interacts with the centrosomal protein γ-tubulin (Thompson et al, 2004). In addition, the β2-adaptin subunit of the clathrin adaptor AP2 associates, at least in vitro, with a component of the mitotic spindle checkpoint, the kinase BubR1. Although the physiological meaning of this interaction is unknown, it might provide a link between endocytic proteins and the mitotic checkpoint machinery (Cayrol et al, 2002). Of note, two accessory components of clathrin coated pits, epsin and Eps15 are phosphorylated at mitosis and such modification reduces their binding to the α-adaptin subunit of AP2 (Chen et al, 1999). Among the different hypothesis that can be envisioned, one appealing possibility is that mitotic phosphorylation of epsin and Eps15 alters their binding capabilities promoting formation of protein complexes working at mitosis and involving partners distinct from AP2. Importantly, epsin has been shown to facilitate spindle organization independently from its endocytic function by using cell-free spindle assembly assays. In these assays, *Xenopus* egg extracts, lacking the membrane cortex, have been depleted of epsin and reconstituted with purified epsin or with epsin lacking the membrane-bending domain. Only full length epsin was able to rescue the spindle defects demonstrating that the membrane curvature activity of epsin is required for the establishment of spindle morphology independently from endocytosis (Liu & Zheng, 2009). This study nicely extends the concept that endocytic proteins have a role in mitosis distinct from the one exerted during interphase.

Given that endocytosis is active throughout the cell cycle and that, at mitosis, some endocytic proteins are also involved in pathways different from internalization, these molecules might play two distinct functions simultaneously thus coordinating membrane traffic with the execution of mitotic events.

Endocytosis and Exocytosis in Signal Transduction and in Cell Migration 173

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Genetic instability is a driving force in tumourigenesis and it is prompted by alteration in centrosome function and in spindle assembly (Lengauer et al, 1998; Lingle et al, 2002; Orr-Weaver & Weinberg, 1998). Since endocytic proteins participate in the regulation of mitotic events, this could represent a novel, previously unrecognized, link between endocytosis and cancer.
