SSNA1 stabilizes dynamic microtubules and detects microtubule damage

Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability - slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin's severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1's functions on microtubule structures in cells.

The Astrin/SKAP Complex Reduces Friction at the Kinetochore-Microtubule Interface

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation, but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens movement and decreases coordination of metaphase sister kinetochores, and increases tension between them. Using laser ablation to isolate kinetochores bound to polymerizing vs depolymerizing microtubules, we show that without SKAP kinetochores move slower on both polymerizing and depolymerizing microtubules, and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to previously described kinetochore proteins that increase grip on microtubules under force, Astrin-SKAP reduces grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

Mechanical torque promotes bipolarity of the mitotic spindle through multi-centrosomal clustering

Intracellular forces shape cellular organization and function. One example is the mitotic spindle, a cellular machine consisting of multiple chromosomes and centrosomes which interact via dynamic microtubule filaments and motor proteins, resulting in complicated spatially dependent forces. For a cell to divide properly, is important for the spindle to be bipolar, with chromosomes at the center and multiple centrosomes clustered into two 'poles' at opposite sides of the chromosomes. Experimental observations show that in unhealthy cells, the spindle can take on a variety of patterns. What forces drive each of these patterns? It is known that attraction between centrosomes is key to bipolarity, but what the prevents the centrosomes from collapsing into a monopolar configuration? Here, we explore the hypothesis that torque rotating chromosome arms into orientations perpendicular to the centrosome-centromere vector promotes spindle bipolarity. To test this hypothesis, we construct a pairwise-interaction model of the spindle. On a continuum version of the model, an integro-PDE system, we perform linear stability analysis and construct numerical solutions which display a variety of spatial patterns. We also simulate a discrete particle model resulting in a phase diagram that confirms that the spindle bipolarity emerges most robustly with torque. Altogether, our results suggest that rotational forces may play an important role in dictating spindle patterning.

Spatiotemporal coordination of the RSF1-PLK1-Aurora B cascade establishes mitotic signaling platforms

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.

A liquid +TIP-network drives microtubule dynamics through tubulin condensation

Tubulin dimers assemble into a dynamic microtubule network throughout the cell. Microtubule dynamics and network organization must be precisely tuned for the microtubule cytoskeleton to regulate a dazzling array of dynamic cell behaviors. Since tubulin concentration determines microtubule growth, we studied here a novel regulatory mechanism of microtubule dynamics: local tubulin condensation. We discovered that two microtubule tip-binding proteins, CLIP-170 and EB3, undergo phase separation and form an EB3/CLIP-170 droplet at the growing microtubule tip. We prove that this +TIP-droplet has the capacity to locally condense tubulin. This process of tubulin co-condensation is spatially initiated at the microtubule tip and temporally regulated to occur only when there is tip growth. Tubulin condensation at the growing microtubule tip increases growth speeds three-fold and strongly reduces depolymerization events. With this work we establish a new mechanism to regulate microtubule dynamics by enrichment of tubulin at strategically important locations: the growing microtubule tips.

KIF2C regulates synaptic plasticity and cognition by mediating dynamic microtubule invasion of dendritic spines

Dynamic microtubules play a critical role in cell structure and function. In nervous system, microtubules specially extend into and out of synapses to regulate synaptic development and plasticity. However, the detailed polymerization especially the depolymerization mechanism that regulates dynamic microtubules in synapses is still unclear. In this study, we find that KIF2C, a dynamic microtubule depolymerization protein without known function in the nervous system, plays a vital role in the structural and functional plasticity of synapses and regulates cognitive function. Using RNAi knockdown and conditional knockout approaches, we showed that KIF2C regulates spine morphology and synaptic membrane expression of AMPA (α- amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Moreover, KIF2C deficiency leads to impaired excitatory transmission, long-term potentiation, and altered cognitive behaviors in mice. Mechanistically, KIF2C regulates microtubule dynamics and microtubule invasion of spines in neurons by its microtubule depolymerization capability in a neuronal activity-dependent manner. This study explores a novel function of KIF2C in the nervous system and provides an important regulatory mechanism on how microtubule invasion of spines regulates synaptic plasticity and cognition behaviors.

An anchoring complex recruits katanin for microtubule severing at the plant cortical nucleation sites

Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei

In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B.

Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte

The microtubule network of the cardiomyocyte exhibits specialized architecture, stability and mechanical behavior that accommodate the demands of working muscle cells. Stable, post-translationally detyrosinated microtubules are physical coupled to the sarcomere, the contractile apparatus of muscle, and resist sarcomere motion to regulate muscle mechanics and mechanosignaling. Control of microtubule growth and shrinkage dynamics represents a potential intermediate in the formation of a stable, physically coupled microtubule network, yet the molecular determinants that govern dynamics are unknown. Here we test the hypothesis that desmin intermediate filaments may stabilize growing microtubules at the sarcomere Z-disk in a detyrosination-dependent manner. Using a combination of biochemical assays and direct observation of microtubule plus-end dynamics in primary adult cardiomyocytes, we determine that: 1) tyrosination increases the frequency of microtubule depolymerization and reduces the pausing of microtubules at the Z-disk, leading to a more dynamic microtubule; and 2) desmin intermediate filaments stabilize both growing and shrinking microtubules specifically at the Z-disk and protect them from depolymerization. This stabilizes iteratively growing, detyrosinated microtubules between adjacent sarcomeres, which promotes the formation of high-energy microtubules that buckle between sarcomeres and elevates myocyte viscoelasticity. Our findings inform on how the tubulin code and intermediate filaments regulate microtubule dynamics, and provide mechanism to the establishment of a spatially organized, physically coupled, and long-lived microtubule network in the cardiomyocyte.

SSNA1 stabilizes dynamic microtubules and detects microtubule damage

ABSTRACTSjögren’s Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. We find that SSNA1 modulates all parameters of microtubule dynamic instability – slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue. SSNA1 accumulation on dynamic microtubule ends correlates with the growth rate slow-down. Furthermore, SSNA1 prevents catastrophe when soluble tubulin is removed or sequestered by Op18/Stathmin. Finally, SSNA1 detects spastin-induced damage and inhibits spastin’s severing activity. Therefore, SSNA1 is both a potent microtubule stabilizing protein and a sensor of microtubule damage; activities that likely underlie SSNA1’s cellular functions.