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.

Microtubules are indispensable for Golgi complex assembly and maintenance that is an integral part of cytoplasm organization in interphase mammalian cells. Here, we show that two discrete microtubule subsets drive two distinct, yet simultaneous, stages of Golgi assembly. In addition to the radial centrosomal microtubule array, which positions the Golgi in the cell center, we identify a role for microtubules that form at the Golgi membranes in a manner dependent on microtubule regulators CLASPs. These Golgi-derived microtubules draw Golgi mini-stacks together in tangential fashion and are critical for establishing continuity and proper morphology of the Golgi complex. We propose that specialized functions of these two microtubule arrays arise from their specific geometries. Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs suggesting that correct organization of the Golgi complex by microtubules is essential for cell polarization and motility.

The actin cytoskeleton at adherens junctions acts as a dynamic push–pull system, wherein pushing forces generated in both contacting cells through Arp2/3 complex–dependent actin polymerization maintain cadherin transinteraction in the extracellular gap, whereas pulling forces stabilize intercellular adhesion complexes.

The kinesin KIF1C is known to regulate podosomes, actin-rich adhesion structures, which remodel the extracellular matrix during physiological processes. Here we show that KIF1C is a player in the podosome-inducing signaling cascade. Upon induction of podosome formation by protein kinase C, KIF1C translocation to the cell periphery intensifies and KIF1C accumulates in the proximity of peripheral microtubules enriched with plus tip-associated proteins CLASPs and around podosomes. Importantly, without CLASPs, both KIF1C trafficking and podosome formation are suppressed. Moreover, chimeric mitochondria-targeted CLASP2 recruits KIF1C, suggesting a transient CLASP-KIF1C association. We propose that CLASP creates preferred microtubule tracks for KIF1C to promote podosome induction downstream of PKC.

Dendritic spines are postsynaptic structures in neurons often having a mushroom-like shape. Physiological significance and cytoskeletal mechanisms that maintain this shape are poorly understood. The spectrin-based membrane skeleton maintains the biconcave shape of erythrocytes,butwhetherspectrinsalsodeterminetheshapeofnonerythroidcellsislessclear.Weshowthat␤IIIspectrininhippocampalandcortical neurons from rodent embryos of both sexes is distributed throughout the somatodendritic compartment but is particularly enriched in the neck and base of dendritic spines and largely absent from spine heads. Electron microscopy revealed that ␤III spectrin forms a detergent-resistant cytoskeletal network at these sites. Knockdown of ␤III spectrin results in a significant decrease in the density of dendritic spines. Surprisingly, the density of presynaptic terminals is not affected by ␤III spectrin knockdown. However, instead of making normal spiny synapses, the presynaptic structures in ␤III spectrin-depleted neurons make shaft synapses that exhibit increased amplitudes of miniature EPSCs indicative of excessive postsynaptic excitation. Thus, ␤III spectrin is necessary for formation of the constricted shape of the spine neck, which in turn controls communication between the synapse and the parent dendrite to prevent excessive excitation. Notably, mutations of SPTNB2 encoding ␤III spectrin are associated with neurodegenerative syndromes, spinocerebellar ataxia Type 5, and spectrin-associated autosomal recessive cerebellarataxiaType1,butmolecularmechanismslinking␤IIIspectrinfunctionstoneuronalpathologiesremainunresolved.Ourdatasuggest that spinocerebellar ataxia Type 5 and spectrin-associated autosomal recessive cerebellar ataxia Type 1 pathology likely arises from poorly controlled synaptic activity that leads to excitotoxicity and neurodegeneration.