The large Nuclear Mitotic Apparatus (NuMA) protein is an abundant component of interphase nuclei and an essential player in mitotic spindle assembly and maintenance. With its partner, cytoplasmic dynein, NuMA uses its cross-linking properties to tether microtubules to spindle poles. NuMA and its invertebrate homologues play a similar tethering role at the cell cortex, thereby mediating essential asymmetric divisions during development. Despite its maintenance as a nuclear component for decades after the final mitosis of many cell types (including neurons), an interphase role for NuMA remains to be established, although its structural properties implicate it as a component of a nuclear scaffold, perhaps as a central constituent of the proposed nuclear matrix.

The Golgi apparatus is a network of polarized cisternae localized to the perinuclear region in mammalian cells. It undergoes extensive vesiculation at the onset of mitosis and its reassembly requires factors that are in part segregated via the mitotic spindle. Here we show that unlike typical Golgi markers, the Golgiprotein p115 partitioned with the spindle poles throughout mitosis. An armadillo-fold in its N terminus mediated a novel interaction between p115 and ␥-tubulin and functioned in its centrosomal targeting. Both the N-and C-terminal regions of p115 were required to maintain Golgi structure. Strikingly, p115 was essential for mitotic spindle function and the resolution of the cytokinetic bridge because its depletion resulted in spindle collapse, chromosome missegregation, and failed cytokinesis. We demonstrate that p115 plays a critical role in mitosis progression, implicating it as the only known golgin to regulate both mitosis and apoptosis.

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin. INTRODUCTIONThe Golgi apparatus is a polarized organelle that in interphase cells exists as a highly organized membranous network in the juxtanuclear region of the cell; it plays key roles in protein and lipid modification as well as cargo sorting. The structural organization of the Golgi apparatus is maintained by a family of high-molecular-weight proteins (e.g., GM130, GRASP65, Golgin-160, and p115), cytoskeletal components such as ␤III-spectrin as well as by the microtubule cytoskeleton (Nakamura et al., 1995;Barr et al., 1997;Stankewich et al., 1998;Shorter and Warren, 1999;Thyberg and Moskalewski, 1999). Several lipids, including phosphatidic acid (PA), diacylglycerol, phosphatidylinositol 4-phosphate [PtdIns(4)P], phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] have also been implicated in maintaining Golgi structure and function (De Matteis et al., 2005). In mammalian cells, the Golgi apparatus has a characteristic lace-like morphology that disassembles into vesicles and tubules at the onset of mitosis and reforms in telophase after the equal distribution of Golgi-derived vesicles into daughter cells. In contrast, during apoptosis the organelle fragments irreversibly (Machamer, 2003). Two models have been proposed to explain the inheritance of the Golgi complex. One suggests that free mitotic tubulovesicular clusters (MGCs) are the unit of Golgi inheritance (Jokitalo et al., 2001;Axelsson and Warren, 2004), whereas a second model proposes that the Golgi is inherited through a merged endoplasmic reticulum (ER)/Golgi compartment (Zaal et al., 1999). Furthermore, Seemann et al. (2002) suggested that the Golgi matrix provides a scaffold for reformation of the membranous ...

During the G2-M transition, the highly organized Golgi apparatus undergoes reversible fragmentation through unstacking of the cisternal ribbon and disassembly into radially dispersed vesicles and tubules. These Golgi-derived fragments redistribute randomly within the cytoplasm, partition stochastically, and in telophase coalesce to generate a functionally and structurally intact Golgi complex. Here we identified a novel step in postmitotic Golgi reassembly that requires the clathrin heavy chain (CHC). We used siRNA-mediated CHC knockdown, biochemistry, and morphological analysis and showed that the spindle- and spindle pole-associated clathrin pools are membrane-bound and required for postmitotic Golgi reassembly. The results presented here show that clathrin remains associated with the spindle poles throughout mitosis and that this clathrin pool is distinct from the previously characterized spindle-associated population. We suggest that clathrin may provide a template for postmitotic Golgi reassembly and cisternal remodeling. In absence of the CHC, the Golgi apparatus remained disconnected and disordered and failed to regain its characteristic perinuclear, lace-like morphology. Our findings build on previous independent reports that clathrin is required for Golgi reassembly following disruption with pharmacological agents and for mitotic chromosome congression.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) synthesis has been implicated in maintaining the function of the Golgi apparatus. Here we demonstrate that the inhibition of PtdIns(4,5)P 2 synthesis in vitro in response to primary alcohol treatment and the kinetics of Golgi fragmentation in vivo were very rapid and tightly coupled. Preloading Golgi membranes with short chain phosphatidic acid abrogated the alcohol-mediated inhibition of PtdIns(4,5)P 2 synthesis in vitro. We also show that fragmentation of the Golgi apparatus in response to diminished PtdIns(4,5)P 2 synthesis correlated with both the phosphorylation of a Golgi form of ␤III spectrin, a PtdIns(4,5)P 2 -interacting protein, and changes in its intracellular redistribution. The data are consistent with a model suggesting that the decreased PtdIns(4,5)P 2 synthesis and the phosphorylation state of ␤III spectrin modulate the structural integrity of the Golgi apparatus.