Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.

The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.

Spastin regulates anaphase chromosome separation distance and microtubule-containing nuclear tunnels.

Nuclear envelope reassembly during the final stages of each mitosis depends on disassembling spindle microtubules without disrupting chromosome separation. This process involves the transient recruitment of the ESCRT-III complex and spastin, a microtubule-severing AAA (ATPases associated with diverse cellular activities) mechanoenzyme, to late-anaphase chromosomes. However, dissecting mechanisms underlying these rapid processes, which can be completed within minutes, has been difficult.

Structure of the γ-tubulin ring complex-capped microtubule.

Unlabelled: Microtubules are composed of α/β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the filament's long-axis. We report that the human ∼2.

A nucleotide binding-independent role for γ-tubulin in microtubule capping and cell division.

The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC's capping activity.

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function.

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB).

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase.

MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during Anaphase.

During cytokinesis, signals from the anaphase spindle direct the formation and position of a contractile ring at the cell cortex [1]. The chromosomal passenger complex (CPC) participates in cytokinesis initiation by signaling from the spindle midzone and equatorial cortex [2], but the mechanisms underlying the anaphase-specific CPC localization are currently unresolved. Accumulation of the CPC at these sites requires the presence of microtubules and the mitotic kinesin-like protein 2, MKLP2 (KIF20A), a member of the kinesin-6 family [2-7], and this has led to the hypothesis that the CPC is transported along microtubules by MKLP2 [3-5, 7].

CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation.

Microtubules play a key role in cell division, motility, and intracellular trafficking. Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can be induced by mechanical stress, severing enzymes, or occur spontaneously during polymerization [2-6].

Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules.

Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example, tubules of endoplasmic reticulum (ER) can be extended by kinesin- and dynein-mediated transport and through the association with the tips of dynamic microtubules. The binding between ER and growing microtubule plus ends requires End Binding (EB) proteins and the transmembrane protein STIM1, which form a tip-attachment complex (TAC), but it is unknown whether these proteins are sufficient for membrane remodeling.

Feedback-Driven Assembly of the Axon Initial Segment.

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood.

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking.

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy.

CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain.

The dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin.

Tipping microtubule dynamics, one protofilament at a time.

Microtubules are polymeric tubes that switch between phases of growth and shortening, and this property is essential to drive key cellular processes. Microtubules are composed of protofilaments formed by longitudinally arranged tubulin dimers. Microtubule dynamics can be affected by structural perturbations at the plus end, such as end tapering, and targeting only a small subset of protofilaments can alter the dynamics of the whole microtubule.

Septins are involved at the early stages of macroautophagy in .

Autophagy is a conserved cellular degradation pathway wherein double-membrane vesicles called autophagosomes capture long-lived proteins, and damaged or superfluous organelles, and deliver them to the lysosome for degradation. Septins are conserved GTP-binding proteins involved in many cellular processes, including phagocytosis and the autophagy of intracellular bacteria, but no role in general autophagy was known. In budding yeast, septins polymerize into ring-shaped arrays of filaments required for cytokinesis.

Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention.

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors.

The C-terminal tails of heterotrimeric kinesin-2 motor subunits directly bind to α-tubulin1: Possible implications for cilia-specific tubulin entry.

The assembly of microtubule-based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin-2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C-terminal tail fragments of heterotrimeric kinesin-2 and α-tubulin1 isoforms in vitro.

Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth.

Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions.