Convergence of cell cycle regulation and growth factor signals on GRASP65

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.     

GRASP65 controls Golgi position and structure during G2/M transition by regulating the stability of microtubules

The mammalian Golgi apparatus is organized in the form of a ribbon‐like structure positioned near the centrosome. Despite its multimodular organization, the Golgi complex is characterized by a prominent structural plasticity, which is crucial during essential physiological processes, such as the G2 phase of the cell cycle, during which the Golgi ribbon must be “unlinked” into isolated stacks to allow progression into mitosis. Here we show that the Golgi‐associated protein GRASP65, which is well known for its role in Golgi stacking and ribbon formation, is also required for the organization of the microtubule cytoskeleton. GRASP65 is not involved in microtubule nucleation or anchoring. Instead, it is required for the stabilization of newly nucleated microtubules, leading to their acetylation and clustering of Golgi stacks. Ribbon formation and microtubule stabilization are both regulated by JNK/ERK‐mediated phosphorylation of S274 of GRASP65, suggesting that this protein can coordinate the Golgi structure with microtubule organization. In agreement with an important role, tubulin acetylation is strongly reduced during the G2 phase of the cell cycle, allowing the separation of the Golgi stacks. Thus, our data reveal a fundamental role of GRASP65 in the integration of different stimuli to modulate Golgi structure and microtubule organization during cell division.     

Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type ‘GRASP'' domains. Fas/CD95 contains a C-terminal leucine–valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65–Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-X_L as a candidate apoptotic binding partner for GRASP65.     

Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.     

GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis‐trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI‐based retrograde transport vesicles, thus concentrating them in the trans‐Golgi. In genome‐edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis‐Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.     

Classical cadherins control nucleus and centrosome position and cell polarity

Control of cell polarity is crucial during tissue morphogenesis and renewal, and depends on spatial cues provided by the extracellular environment. Using micropatterned substrates to impose reproducible cell–cell interactions, we show that in the absence of other polarizing cues, cell–cell contacts are the main regulator of nucleus and centrosome positioning, and intracellular polarized organization. In a variety of cell types, including astrocytes, epithelial cells, and endothelial cells, calcium-dependent cadherin-mediated cell–cell interactions induce nucleus and centrosome off-centering toward cell–cell contacts, and promote orientation of the nucleus–centrosome axis toward free cell edges. Nucleus and centrosome off-centering is controlled by N-cadherin through the regulation of cell interactions with the extracellular matrix, whereas the orientation of the nucleus–centrosome axis is determined by the geometry of N-cadherin–mediated contacts. Our results demonstrate that in addition to the specific function of E-cadherin in regulating baso-apical epithelial polarity, classical cadherins control cell polarization in otherwise nonpolarized cells.     

The Role of GRASP65 in Golgi Cisternal Stacking and Cell Cycle Progression

In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans‐oligomers. These conclusions, however, require further confirmation in the cell. In this study, we showed that the first 112 amino acids at the N‐terminus (including the first PDZ domain, PDZ1) of the protein are sufficient for oligomerization. Systematic electron microscopic analysis showed that the expression of non‐regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. These results provided evidence and a molecular mechanism by which GRASP65 stacks Golgi cisternal membranes. Further experiments revealed that inhibition of mitotic Golgi disassembly by expressing non‐regulatable GRASP65 mutants did not affect equal partitioning of the Golgi membranes into the daughter cells. However, it delayed mitotic entry and suppressed cell growth; this effect was diminished by dispersing the Golgi apparatus with Brefeldin A treatment prior to mitosis, suggesting that Golgi disassembly at the onset of mitosis plays a role in cell cycle progression.     

GRASP55 regulates Golgi ribbon formation

Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression.     

A direct role for GRASP65 as a mitotically regulated Golgi stacking factor

Cell-free assays that mimic the disassembly and reassembly cycle of the Golgi apparatus during mitosis implicated GRASP65 as a mitotically regulated stacking factor. We now present evidence that GRASP65 is directly involved in stacking Golgi cisternae. GRASP65 is the major phosphorylation target in rat liver Golgi membranes of two mitotic kinases, cdc2-cyclin B and polo-like kinases, which alone will unstack Golgi membranes, generating single cisternae. Mitotic cells microinjected with antibodies to GRASP65 fail to form proper Golgi stacks after cell division. Beads coated with GRASP65 homodimers form extensive aggregates consistent with the formation of trans oligomers. These can be disaggregated using purified cdc2-cyclin B1 and polo-like kinases, and re-aggregated after dephosphorylation of GRASP65. Together, these data demonstrate that GRASP65 has the properties required to bind surfaces together in a mitotically regulated manner.     

Cadherin-2 Controls Directional Chain Migration of Cerebellar Granule Neurons

Long distance migration of differentiating granule cells from the cerebellar upper rhombic lip has been reported in many vertebrates. However, the knowledge about the subcellular dynamics and molecular mechanisms regulating directional neuronal migration in vivo is just beginning to emerge. Here we show by time-lapse imaging in live zebrafish (Danio rerio) embryos that cerebellar granule cells migrate in chain-like structures in a homotypic glia-independent manner. Temporal rescue of zebrafish Cadherin-2 mutants reveals a direct role for this adhesion molecule in mediating chain formation and coherent migratory behavior of granule cells. In addition, Cadherin-2 maintains the orientation of cell polarization in direction of migration, whereas in Cadherin-2 mutant granule cells the site of leading edge formation and centrosome positioning is randomized. Thus, the lack of adhesion leads to impaired directional migration with a mispositioning of Cadherin-2 deficient granule cells as a consequence. Furthermore, these cells fail to differentiate properly into mature granule neurons. In vivo imaging of Cadherin-2 localization revealed the dynamics of this adhesion molecule during cell locomotion. Cadherin-2 concentrates transiently at the front of granule cells during the initiation of individual migratory steps by intramembraneous transport. The presence of Cadherin-2 in the leading edge corresponds to the observed centrosome orientation in direction of migration. Our results indicate that Cadherin-2 plays a key role during zebrafish granule cell migration by continuously coordinating cell-cell contacts and cell polarity through the remodeling of adherens junctions. As Cadherin-containing adherens junctions have been shown to be connected via microtubule fibers with the centrosome, our results offer an explanation for the mechanism of leading edge and centrosome positioning during nucleokinetic migration of many vertebrate neuronal populations.     

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.     

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.     

GRASPing for consensus about the Golgi apparatus

Cisternae of the Golgi apparatus adhere to each other to form stacks, which are aligned side by side to form the Golgi ribbon. Two proteins, GRASP65 and GRASP55, previously implicated in stacking of cisternae, are shown to be required for the formation of the Golgi ribbon.

IntroductionThe Golgi apparatus is an intermediate organelle along the secretory pathway that receives proteins and lipids (“cargo”) from the endoplasmic reticulum, covalently modifies them, and then exports them via transport vesicles for trafficking to the plasma membrane or other organelles. In most eukaryotic cells, disc-shaped membrane cisternae, each containing a distinct repertoire of cargo-processing enzymes, are stacked one on top of another to form the “Golgi stack,” a visual hallmark of the organelle (Fig. 1). The cisternae of the Golgi stack are polarized, with the compartment receiving endoplasmic reticulum–derived cargo termed the cis cisterna followed by the medial; trans; and finally, the trans-Golgi network. The physiological advantages conferred by stacking of Golgi cisternae are unclear, but it is thought to enhance the efficiencies of the sequential chemical modifications of glycoproteins and glycolipids during secretion. Cultured mammalian cells may possess more than 100 Golgi stacks, which are aligned side by side about the centrosome to form the “Golgi ribbon” (Fig. 1). Vesicles and tubules span the intervening, “noncompact” zones between stacks of cisternae, connecting analogous cisternae across the ribbon and thereby ensuring a homogeneous distribution of Golgi resident proteins among all cisternae. During mitosis, the Golgi ribbon is unlinked, the stacks are disassembled, and the cisternae are converted to vesicles and tubules; after cytokinesis, the process is reversed, and the Golgi is rebuilt. The dynamic nature of Golgi structure in interphase and mitotic cells implies the existence of a reversible mechanism that tethers Golgi cisternae to each other to form the stack and a mechanism that aligns and links the stacks into the ribbon.Open in a separate windowFigure 1.The organization of the Golgi apparatus in vertebrate cells. Individual stacks of Golgi cisternae are aligned side to side to form the Golgi ribbon. The GRASP65 and GRASP55 proteins are depicted to be enriched on the rims of the indicated cisternae within individual stacks of cisternae, where they are required to maintain the arrangement of stacks into the ribbon.GRASP proteins tether Golgi cisternae in vitroInvestigations into the molecular basis of Golgi cisterna stacking have ultimately focused attention on a handful of cytoplasmic proteins called “Golgins” and “GRASPs” that are associated with specific Golgi cisternae and interact with each other. Of particular interest are two related proteins GRASP65 and GRASP55 (respective systematic names GORASP1 and GORASP2), discovered by Warren and colleagues via in vitro reconstitution experiments, as capable of mediating stacking of Golgi cisternae (Barr et al., 1997; Shorter et al., 1999). Whereas GRASP65 localizes to the cis cisterna, GRASP55 localization favors medial/trans Golgi cisternae (Shorter et al., 1999); hence, these proteins could, in principle, tether cisternae to form a minimal Golgi stack. In these in vitro assays, perturbations (mutations, antibody interference) to either GRASP65 or GRASP55 inhibited stacking of reformed Golgi cisternae. Moreover, GRASP proteins are phosphorylated in mitosis just before vesiculation of Golgi cisternae, and preventing phosphorylation impairs the disassembly of the Golgi apparatus and mitotic progression (Wang et al., 2003). These findings underpin models of the Golgi stack where GRASP65 and GRASP55, along with Golgin proteins, constitute the core components of a cytoplasmic “matrix” of proteins that surround the cisternae, mediating their stacking as well as the tethering of transport vesicles to cisternae. Curiously, plant cells contain stacked Golgi cisternae, yet they do not express any GRASP or GRASP-related proteins. And some nonvertebrate organisms with stacked Golgi cisternae express just one GRASP-related protein, while the Golgi cisternae are not stacked in other nonvertebrate organisms (e.g., yeast) that express a single GRASP (Glick and Malhotra, 1998). Apparently, the presence or number of GRASP proteins expressed does not correlate with stacked cisternae.Whereas the results of in vitro biochemical assays underpin our conceptions of GRASP protein function, probing their roles in vivo has proven to be quite challenging. First, depletion/deletion of each individual GRASP protein is largely without effect on Golgi stack or ribbon formation, but a very complex phenotype results from depletion/deletion of both GRASP proteins. Thus, some reports conclude that the GRASP proteins function redundantly to stack cisternae (Bekier et al., 2017), while others conclude that the Golgi ribbon, not the stack per se, is perturbed upon loss of GRASP proteins (Puthenveedu et al., 2006; Feinstein and Linstedt, 2008; Xiang and Wang, 2010; Lee et al., 2014; Veenendaal et al., 2014). Recently, two papers published in the Journal of Cell Biology employed different methodologies to perturb GRASP protein functions in vivo (Grond et al., 2020; Zhang and Seemann, 2021), providing the most conclusive insight to date into the roles of GRASP proteins in Golgi structure.The Golgi ribbon is unlinked upon loss of GRASP proteinsRabouille and colleagues used traditional mouse gene knockout technology to delete GRASP65, finding that such mice are viable with no apparent physiological deficits or gross morphological perturbations of the Golgi (Veenendaal et al., 2014). In their recent study (Grond et al., 2020), GRASP55 was deleted in the GRASP65 null background, but double-knockout mice could not be obtained, consistent with GRASP proteins being at least partially physiologically redundant. Next, using a conditional knockout approach, double GRASP null cells were produced postnatally in the small intestine, and the Golgi of intestinal epithelial cells was examined. In these cells, stacked Golgi cisternae were observed, but their arrangement into a ribbon was compromised, a result corroborated by more detailed analysis of cells in organoid cultures. These findings are at odds with the conclusions of Wang and colleagues (Bekier et al., 2017), who used CRISPR-Cas9 gene editing technology to construct cultured mammalian cell lines that do not express GRASP65 and GRASP55. They found that the appearance of Golgi cisternae was grossly altered, resembling clusters of tubules and vesicles (“tubulovesicular clusters”) about swollen cisterna remnants that debatably appeared to be stacked. One possible reason for the disparities between these two studies is that Bekier et al. (2017) documented that loss of GRASP proteins in cultured mammalian cells also resulted in depletion of a subset of Golgin proteins (e.g., GM130, Golgin-45) from Golgi cisternae, so it was not possible to parse the specific contributions of GRASP proteins to Golgi structure.Analyses of siRNA-depleted and gene-edited cell lines and modified animals are often complicated by incomplete depletion of a query protein, unintended loss of other proteins, or compensatory processes that obscure loss-of-function effects. Notably, siRNA depletion of GM130, which is associated with GRASP65 on the cis cisterna, impairs secretory traffic from the endoplasmic reticulum to the Golgi apparatus, resulting in a reduction in the size of Golgi cisternae and diminished interstack connectivity possibly due to vesiculation of cisternae (Seemann et al., 2000; Puthenveedu et al., 2006). To minimize these drawbacks, Zhang and Seemann (2021) used gene editing to modify the GRASP65 and GRASP55 loci to append an inducible protein degradation domain to each protein in cultured mammalian cells, which was used to elicit degradation of the GRASP proteins within just 2 h. Hence, the acute effects of GRASP protein depletion could be determined before the onset of potentially confounding effects. Fluorescence recovery after photobleaching assays of a fluorescently tagged Golgi resident protein revealed that acute depletion of both GRASP65 and GRASP55 resulted in decreased mobility of the resident Golgi enzyme within the ribbon, indicating that connectivity of cisternae between stacks was compromised. Stacks of Golgi cisternae with proper cis–trans polarity were observed by electron and light microscopy, both shortly (∼2 h) after GRASP protein turnover was initiated, and after mitosis, indicating that GRASP proteins are not required to establish or to maintain the Golgi stacks. Importantly, the authors observed no changes in the levels of GRASP-associated proteins (e.g., GM130) when assayed shortly after initiating GRASP protein turnover, but the amounts of several GRASP-associated proteins were reduced after prolonged growth in the absence of GRASP proteins. The results are in general agreement with experiments by Jarvela and Linstedt (2014), who expressed GRASP65 and GRASP55 fusion proteins appended with “killer RFP” and used chomophore-assisted light inactivation to rapidly (1 min) ablate the proteins in cultured mammalian cells. Similar to Zhang and Seemann (2021), they observed that the Golgi ribbon was disassembled upon inactivation of GRASP proteins, but stacking of cisternae was unaffected. Taken together, these results conclusively show that acute depletion of GRASP65 and GRASP55 impairs lateral linking of stacked Golgi cisternae within the ribbon while not affecting stacking of cisternae.Conclusions and perspectivesA body of work now more than 20 years old has shown that GRASP65 and GRAPS55 are core structural components of a matrix of cytoplasmic proteins associated with Golgi cisternae; however, the Grond et al. (2020) and Zhang and Seemann (2021) reports now firmly establish that GRASP proteins are dispensable for stacking of Golgi cisterna and indicate that they are required for linking Golgi stacks within the ribbon. These new studies suggest that the integrity of the Golgi matrix critically depends on the presence of GRASP proteins, and their absence perturbs the balance of cargo flow through the Golgi, reducing the interstack exchange required to maintain connectivity of stacks within the ribbon. How might GRASP proteins facilitate linking of stacks within the Golgi ribbon? When the ribbon is disrupted (using the microtubule depolymerizing reagent nocodazole) and individual Golgi stacks are examined, GRASP65 and GRASP55 appear to be enriched at the rims of Golgi cisternae (Fig. 1; Tie et al., 2018). Hence, the GRASP proteins are positioned at the vesicle-rich interface between adjacent cisternal stacks. Grond et al. (2020) observed reductions in the size of Golgi cisternae in cells deleted of both GRASP proteins and speculated that this may be due to increased coatomer I vesicle formation at the rims of cisternae. In this view, GRASP proteins dampen vesicle flux at the rims of Golgi cisternae, a model supported by the observation that depletion of GRASP proteins leads to an increase in secretion rate (Wang et al., 2008). These new studies firmly shift our view of GRASP protein function away from the stacking of Golgi cisternae, and we look forward to new mechanistic insights into the roles of GRASP proteins in Golgi ribbon formation as well as in non–Golgi-dependent processes, such as unconventional protein secretion (Kinseth et al., 2007).     

Mitotic Phosphorylation of Golgi Reassembly Stacking Protein 55 by Mitogen-activated Protein Kinase ERK2

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Excess centrosomes disrupt endothelial cell migration via centrosome scattering

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome–MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.     

Allosteric regulation of GRASP protein-dependent Golgi membrane tethering by mitotic phosphorylation

Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.     

Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.     

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.     

Golgi apparatus fragmentation participates in oxidized low-density lipoprotein-induced endothelial cell injury

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial injury plays crucial roles in the development of arteriosclerosis (AS). Golgi apparatus (GA) fragmentation is involved in various pathological processes, including endothelial injury. However, the role of GA fragmentation in ox-LDL-induced endothelial injury has not been determined. In this study, human umbilical vein endothelial cells (HUVECs) subjected to ox-LDL were used as an in vitro AS model. Herein, we showed that ox-LDL restrained proliferation and induced apoptosis and GA fragmentation of HUVECs. Moreover, overexpression of GRASP65 significantly prevented ox-LDL-induced GA fragmentation and endothelial cell injury by enhancing cell viability, nitric oxide production, and endothelial NOS expression and reducing apoptosis. Mechanistically, ox-LDL resulted in the activation of the extracellular signal-regulated kinase (ERK) pathway in HUVECs. Inactivation of the ERK pathway by U0126 suppressed the phosphorylation of GRASP65, GA fragmentation, and endothelial cell injury induced by ox-LDL. In conclusion, ox-LDL triggers GA fragmentation in HUVECs via activating the ERK signaling pathway, which participates in endothelial injury during the development of AS.