Proline-rich transmembrane protein 2 (PRRT2) regulates the actin cytoskeleton during synaptogenesis.

Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.

Aurora-A recruitment and centrosomal maturation are regulated by a Golgi-activated pool of Src during G2.

The Golgi apparatus is composed of stacks of cisternae laterally connected by tubules to form a ribbon-like structure. At the onset of mitosis, the Golgi ribbon is broken down into discrete stacks, which then undergo further fragmentation. This ribbon cleavage is required for G2/M transition, which thus indicates that a 'Golgi mitotic checkpoint' couples Golgi inheritance with cell cycle transition. We previously showed that the Golgi-checkpoint regulates the centrosomal recruitment of the mitotic kinase Aurora-A; however, how the Golgi unlinking regulates this recruitment was unknown. Here we show that, in G2, Aurora-A recruitment is promoted by activated Src at the Golgi. Our data provide evidence that Src and Aurora-A interact upon Golgi ribbon fragmentation; Src phosphorylates Aurora-A at tyrosine 148 and this specific phosphorylation is required for Aurora-A localization at the centrosomes. This process, pivotal for centrosome maturation, is a fundamental prerequisite for proper spindle formation and chromosome segregation.

PRRT2 Is a Key Component of the Ca(2+)-Dependent Neurotransmitter Release Machinery.

Heterozygous mutations in proline-rich transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders, including epilepsy, kinesigenic dyskinesia, and migraine. Most of the mutations lead to impaired PRRT2 expression, suggesting that loss of PRRT2 function may contribute to pathogenesis. We show that PRRT2 is enriched in presynaptic terminals and that its silencing decreases the number of synapses and increases the number of docked synaptic vesicles at rest. PRRT2-silenced neurons exhibit a severe impairment of synchronous release, attributable to a sharp decrease in release probability and Ca(2+) sensitivity and associated with a marked increase of the asynchronous/synchronous release ratio. PRRT2 interacts with the synaptic proteins SNAP-25 and synaptotagmin 1/2. The results indicate that PRRT2 is intimately connected with the Ca(2+)-sensing machinery and that it plays an important role in the final steps of neurotransmitter release.

JNK2 controls fragmentation of the Golgi complex and the G2/M transition through phosphorylation of GRASP65.

In mammalian cells, the Golgi complex is composed of stacks that are connected by membranous tubules. During G2, the Golgi complex is disassembled into isolated stacks. This process is required for entry into mitosis, indicating that the correct inheritance of the organelle is monitored by a 'Golgi mitotic checkpoint'. However, the regulation and the molecular mechanisms underlying this Golgi disassembly are still poorly understood. Here, we show that JNK2 has a crucial role in the G2-specific separation of the Golgi stacks through phosphorylation of Ser277 of the Golgi-stacking protein GRASP65 (also known as GORASP1). Inhibition of JNK2 by RNA interference or by treatment with three unrelated JNK inhibitors causes a potent and persistent cell cycle block in G2. JNK activity becomes dispensable for mitotic entry if the Golgi complex is disassembled by brefeldin A treatment or by GRASP65 depletion. Finally, measurement of the Golgi fluorescence recovery after photobleaching demonstrates that JNK is required for the cleavage of the tubules connecting Golgi stacks. Our findings reveal that a JNK2-GRASP65 signalling axis has a crucial role in coupling Golgi inheritance and G2/M transition.

Golgi complex fragmentation in G2/M transition: An organelle-based cell-cycle checkpoint.

In mammalian cells, the Golgi complex is organized into a continuous membranous system known as the Golgi ribbon, which is formed by individual Golgi stacks that are laterally connected by tubular bridges. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multistage process that is required for its correct partitioning into the daughter cells. Importantly, inhibition of this Golgi disassembly results in cell-cycle arrest at the G2 stage, suggesting that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Here, we discuss the mechanisms and regulation of the Golgi ribbon breakdown and briefly comment on how Golgi partitioning may inhibit G2/M transition.

The role of Aurora-A kinase in the Golgi-dependent control of mitotic entry.

During mitosis, the Golgi complex undergoes a multi-step fragmentation process that is instrumental to its correct partitioning into the daughter cells. To prepare for this segregation, the Golgi ribbon is initially separated into individual stacks during the G2 phase of the cell cycle. Then, at the onset of mitosis, these individual stacks are further disassembled into dispersed fragments. Inhibition of this Golgi fragmentation step results in a block or delay of G2/M transition, depending on the experimental approach. Thus, correct segregation of the Golgi complex appears to be monitored by a 'Golgi mitotic checkpoint'. Using a microinjection-based approach, we recently identified the first target of the Golgi checkpoint, whereby a block of this Golgi fragmentation impairs recruitment of the mitotic kinase Aurora-A to, and its activation at, the centrosomes. Overexpression of Aurora-A can override this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. We have also shown that this block of Aurora-A recruitment to the centrosomes is not mediated by the known mechanisms of regulation of Aurora-A function. Here we discuss our findings in relation to the known functions of Aurora-A.

Golgi partitioning controls mitotic entry through Aurora-A kinase.

At the onset of mitosis, the Golgi complex undergoes a multistep fragmentation process that is required for its correct partitioning into the daughter cells. Inhibition of this Golgi fragmentation results in cell cycle arrest at the G2 stage, suggesting that correct inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." However, the molecular basis of this G2 block is not known. Here, we show that the G2-specific Golgi fragmentation stage is concomitant with centrosome recruitment and activation of the mitotic kinase Aurora-A, an essential regulator for entry into mitosis. We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression. Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.

Mitotic inheritance of the Golgi complex.

In mammals, the Golgi complex is structured in the form of a continuous membranous system composed of stacks connected by tubular bridges, the "Golgi ribbon". At the onset of mitosis, the Golgi complex undergoes a multi-step fragmentation process that is required for its correct partition into the dividing cells. Regulation of Golgi fragmentation and cell cycle progression appear to be precisely coordinated. Here, we review recent studies that are revealing the fundamental mechanisms, the molecular players and the biological significance of the mitotic inheritance of the Golgi complex in mammalian cells.