Francis A. Barr

Polo-like kinases and the orchestration of cell division.

Polo-like kinases (Plks) are increasingly recognized as key regulators of mitosis, meiosis and cytokinesis. In agreement with a broad range of proposed functions during cell division, Plks are subject to complex temporal and spatial control. Recent findings are uncovering the mechanisms of Plk regulation, notably their targeting to different cellular structures through interactions with phosphorylated docking proteins. Moreover, information is emerging on the substrate specificity of Plks and the role of individual substrates in M-phase progression.

Cytokinesis: Placing and Making the Final Cut

Cytokinesis is the process by which cells physically separate after the duplication and spatial segregation of the genetic material. A number of general principles apply to this process. First the microtubule cytoskeleton plays an important role in the choice and positioning of the division site. Once the site is chosen, the local assembly of the actomyosin contractile ring remodels the plasma membrane. Finally, membrane trafficking to and membrane fusion at the division site cause the physical separation of the daughter cells, a process termed abscission. Here we will discuss recent advances in our understanding of the mechanisms of cytokinesis in animals, yeast, and plants.

GRASP65, A PROTEIN INVOLVED IN THE STACKING OF GOLGI CISTERNAE

NEM prevents mitotic reassembly of Golgi cisternae into stacked structures. The major target of NEM is a 65 kDa protein conserved from yeast to mammals. Antibodies to this protein and a recombinant form of it block cisternal stacking in a cell-free system, justifying its designation as a Golgi ReAssembly Stacking Protein (GRASP65). One of the two minor targets of NEM is GM130, previously implicated in the docking of transport vesicles and mitotic fragmentation of the Golgi stack. GRASP65 is complexed with GM130 and is tightly bound to Golgi membranes, even under mitotic conditions when both are heavily phosphorylated. These results link vesicle docking, stacking of Golgi cisternae, and the disruption of both of these interactions during mitosis.

Rab GEFs and GAPs.

Rabs are GTP-binding proteins with conserved functions in membrane trafficking. They are regulated by a diverse group of structurally unrelated GDP-GTP exchange factors (GEFs), and a family of GTP-hydrolysis activating proteins (GAPs) containing the conserved TBC domain. Recent structural and cell biological studies shed new light on the mechanisms of Rab GEF and GAP action, and the cellular trafficking pathways they act in.

Functional dissection of Rab GTPases involved in primary cilium formation

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

Golgins in the structure and dynamics of the Golgi apparatus

Golgins are a family of coiled-coil proteins associated with the Golgi apparatus necessary for tethering events in membrane fusion and as structural supports for Golgi cisternae. Recent work has shown that golgins such as GM130, golgin-45 and p115 bind to Rab GTPases via their coiled-coil domains, and that GM130, rather than being part of a static structural matrix, is in dynamic exchange between the membrane surface and the cytoplasm. Golgins such as bicaudal-D1 and -D2 bind to Rab6, but, rather than tethering membranes together, link vesicles to the cytoskeleton, thus adding a new function for this class of proteins. Other golgins containing the Golgi targeting GRIP domain, rather than binding Rabs, interact with and are recruited to membranes by another class of GTPase, the Arls. Current evidence therefore suggests that golgins function in a variety of membrane-membrane and membrane-cytoskeleton tethering events at the Golgi apparatus, and that all these are regulated by small GTPases of the Rab and Arl families.

Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2.

Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the central spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B–INCENP complex remains at the centromeres and fails to relocate to the central spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the central spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the central spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.

TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

The noncatalytic RabGAP protein TBC1D14 regulates the Rab11- and ULK1-positive recycling endosomes required for autophagosome formation upon starvation

Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae

The nature of the complex containing GRASP65, a membrane protein involved in establishing the stacked structure of the Golgi apparatus, and GM130, a putative Golgi matrix protein and vesicle docking receptor, was investigated. Gel filtration revealed that GRASP65 and GM130 interact in detergent extracts of Golgi membranes under both interphase and mitotic conditions, and that this complex can bind to the vesicle docking protein p115. Using in vitro translation and site‐directed mutagenesis in conjunction with immunoprecipitation, the binding site for GRASP65 on GM130 was mapped to the sequence xxNDxxxIMVI‐COOH at the C‐terminus of GM130, a region known to be required for its localization to the Golgi apparatus. The same approach was used to show that the binding site for GM130 on GRASP65 maps to amino acids 189–201, a region conserved in the mammalian and yeast proteins and reminiscent of PDZ domains. Using green fluorescent protein (GFP)‐tagged reporter constructs, it was shown that one essential function of the interaction between GRASP65 and GM130 is in the correct targeting of the two proteins to the Golgi apparatus.