Pregabalin improves axon regeneration and motor outcome in a rodent stroke model.

Ischaemic stroke remains a leading cause of death and disability worldwide. Surviving neurons in the peri-infarct area are able to establish novel axonal projections to juxtalesional regions, but this regeneration is curtailed by a growth-inhibitory environment induced by cells such as reactive astrocytes in the glial scar. Here, we found that the astroglial synaptogenic cue thrombospondin-1 is upregulated in the peri-infarct area, and hence tested the effects of the anticonvulsant pregabalin, a blocker of the neuronal thrombospondin-1 receptor Alpha2delta1/2, in a mouse model of cortical stroke. Studying axonal projections after cortical stroke in mice by three-dimensional imaging of cleared whole-brain preparations, we found that pregabalin, when administered systemically for 5 weeks after stroke, augments novel peri-infarct motor cortex projections and improves skilled forelimb motor function. Thus, the promotion of axon elongation across the glial scar by pregabalin represents a promising target beyond the acute phase after stroke to improve structural and functional recovery.

An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury.

Axons in the adult mammalian central nervous system fail to regenerate after spinal cord injury. Neurons lose their capacity to regenerate during development, but the intracellular processes underlying this loss are unclear. We found that critical components of the presynaptic active zone prevent axon regeneration in adult mice. Transcriptomic analysis combined with live-cell imaging revealed that adult primary sensory neurons downregulate molecular constituents of the synapse as they acquire the ability to rapidly grow their axons. Pharmacogenetic reduction of neuronal excitability stimulated axon regeneration after adult spinal cord injury. Genetic gain- and loss-of-function experiments uncovered that essential synaptic vesicle priming proteins of the presynaptic active zone, but not clostridial-toxin-sensitive VAMP-family SNARE proteins, inhibit axon regeneration. Systemic administration of Baclofen reduced voltage-dependent Ca2+ influx in primary sensory neurons and promoted their regeneration after spinal cord injury. These findings indicate that functional presynaptic active zones constitute a major barrier to axon regeneration.

The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation.

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72-γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72-γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72-γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template.

MOZART1/Mzt1 is required for the localization of γ-tubulin complexes to microtubule (MT)-organizing centers from yeast to human cells. Nevertheless, the molecular function of MOZART1/Mzt1 is largely unknown. Taking advantage of the minimal MT nucleation system of Candida albicans, we reconstituted the interactions of Mzt1, γ-tubulin small complex (γ-TuSC), and γ-tubulin complex receptors (γ-TuCRs) Spc72 and Spc110 in vitro. With affinity measurements, domain deletion, and swapping, we show that Spc110 and Mzt1 bind to distinct regions of the γ-TuSC. In contrast, both Mzt1 and γ-TuSC interact with the conserved CM1 motif of Spc110/Spc72. Spc110/Spc72 and Mzt1 constitute "oligomerization chaperones," cooperatively promoting and directing γ-TuSC oligomerization into MT nucleation-competent rings. Consistent with the functions of Mzt1, human MOZART1 directly interacts with the CM1-containing region of the γ-TuCR CEP215. MOZART1 depletion in human cells destabilizes the large γ-tubulin ring complex and abolishes CEP215CM1-induced ectopic MT nucleation. Together, we reveal conserved functions of MOZART1/Mzt1 through interactions with γ-tubulin complex subunits and γ-TuCRs.

Targeting of γ-tubulin complexes to microtubule organizing centers: conservation and divergence.

Organisms with closed or open mitosis have differentially evolved various gamma-tubulin complex (γ-TuC) recruiting factors to organize diverse cellular microtubule (MT) arrays, including the mitotic spindle. γ-TuC recruiting factors not only target the γ-TuC to MT nucleation sites, but also regulate MT nucleation activity by generating the template for MT nucleation or promoting the MT nucleation activity of pre-existing γ-tubulin ring complexes (γ-TuRCs). Here we outline the current understanding of MT nucleator assembly and its regulation by γ-tubulin small complex (γ-TuSC) receptors. Moreover, we discuss the emergence of γ-TuC recruiting factors through evolution with augmented complexity and diversity and propose a hypothesis to account for the evolution of these factors in cooperative spindle assembly.

The γ-tubulin-specific inhibitor gatastatin reveals temporal requirements of microtubule nucleation during the cell cycle.

Inhibitors of microtubule (MT) assembly or dynamics that target α/ß-tubulin are widely exploited in cancer therapy and biological research. However, specific inhibitors of the MT nucleator γ-tubulin that would allow testing temporal functions of γ-tubulin during the cell cycle are yet to be identified. By evolving ß-tubulin-binding drugs we now find that the glaziovianin A derivative gatastatin is a γ-tubulin-specific inhibitor. Gatastatin decreased interphase MT dynamics of human cells without affecting MT number. Gatastatin inhibited assembly of the mitotic spindle in prometaphase. Addition of gatastatin to preformed metaphase spindles altered MT dynamics, reduced the number of growing MTs and shortened spindle length. Furthermore, gatastatin prolonged anaphase duration by affecting anaphase spindle structure, indicating the continuous requirement of MT nucleation during mitosis. Thus, gatastatin facilitates the dissection of the role of γ-tubulin during the cell cycle and reveals the sustained role of γ-tubulin.

Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation.

Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains.DOI: http://dx.doi.org/10.7554/eLife.02208.001.

Molecular mechanisms that restrict yeast centrosome duplication to one event per cell cycle.

BACKGROUND: The spindle pole body (SPB) of budding yeast is the functional equivalent of the mammalian centrosome. Like the centrosome, the SPB duplicates once per cell cycle. The new SPB assembles adjacent to the mother SPB at a substructure called the bridge. The half-bridge, the bridge precursor, is a one-sided extension of the SPB central plaque layered on both sides of the nuclear envelope. Parallel Sfi1 molecules longitudinally span the half-bridge with their N termini embedded in the SPB central plaque, whereas their C termini mark the half-bridge distal end. In early G1, half-bridge elongation by antiparallel C-to-C dimerization of Sfi1 exposes free N-Sfi1 where the new SPB assembles. After SPB duplication, the dimerized Sfi1 is severed to allow spindle formation and SPB reduplication. RESULTS: We show that Sfi1 C-terminal domain harbors phosphorylation sites for Cdk1 and the polo-like kinase Cdc5. Cdk1 and, to a lesser extent, Cdc5 inhibit SPB duplication as phosphomimetic sfi1 mutations lead to metaphase cells with a single SPB. In contrast, phosphoinhibitory sfi1 mutations in Cdk1 sites are lethal because cells fail to sever the bridge after SPB duplication. Moreover, Cdc14 dephosphorylates C-Sfi1 to prepare it for a new round of duplication, and the kinase Mps1 promotes Sfi1 extension in G1. CONCLUSIONS: Positive (Cdc14) and negative (Cdk1 and Cdc5) SPB duplication signals are integrated at the level of the half-bridge component Sfi1. In addition, Mps1 activates Sfi1 duplication. Fluctuating activities of these regulators ensure one SPB duplication event per cell cycle.

Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function.

The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human.