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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. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division., Journal of Cell Biology, 222 (3), ISSN:0021-9525, ISSN:1540-8140

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). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubulenucleating “core” in the holocomplex., The Journal of Cell Biology, 220 (3), ISSN:0021-9525, ISSN:1540-8140

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MDa assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge”. The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP), a ∼35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex, γ-TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that γ-TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native γ-TuRCs. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCmini-GFP adopts a partial cone shape presenting only 8-10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the γ-TuRC.

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. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

SummaryMicrotubule network plays 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 either be induced by mechanical stress or occur spontaneously during polymerization [2–4]. Tubulin incorporation can restore microtubule integrity; moreover, “islands” of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3–7]. Microtubule repair occurs in vitro in the presence of tubulin alone [2–4, 8]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASP is an interesting candidate for microtubule repair, because it induces microtubule nucleation, stimulates rescue and suppresses catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [9–16]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2α indeed stimulates microtubule lattice repair. CLASP2α promoted tubulin incorporation into damaged lattice sites thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic flow-induced bending. A single CLASP2α domain, TOG2, which suppresses catastrophes when tethered to microtubules, was sufficient to stimulate microtubule repair, indicating that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity.

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. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.

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. Our findings indicate not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst which are dispensable for autophagic function do not associate with the autophagy specific subcomplex of exocyst.

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. In an unbiased genetic screen and in subsequent targeted analysis, we found autophagy defects in septin mutants. Upon autophagy induction, pre-assembled septin complexes relocalized to the pre-autophagosomal structure (PAS) where they formed non-canonical septin rings at PAS. Septins also colocalized with autophagosomes, where they physically interacted with the autophagy proteins Atg8 and Atg9. When autophagosome degradation was blocked in septin-mutant cells, fewer autophagic structures accumulated, and an autophagy mutant defective in early stages of autophagosome biogenesis (atg1Δ), displayed decreased septin localization to the PAS. Our findings support a role for septins in the early stages of budding yeast autophagy, during autophagosome formation. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Septin proteins, which form cytoskeletal filaments and bind membranes, are required for efficient assembly of functional autophagic structures in budding yeast cells.

Autophagy is a conserved cellular degradation pathway wherein a double membrane vesicle, called as an autophagosome captures longlived proteins, damaged or superfluous organelles and delivers to the lysosome for degradation1. We have identified a novel role for septins in autophagy. Septins are GTP-binding proteins that localize at the bud-neck and are involved in cytokinesis in budding yeast2. We show that septins under autophagy prevalent conditions are majorly localized to the cytoplasm in the form of punctate structures. Further, we report that septins not only localize to pre-autophagosomal structure (PAS) but also to autophagosomes in the form of punctate structures. Interestingly, septins also form small non-canonical rings around PAS during autophagy. Furthermore, we observed that in one of the septin Ts" mutant,cdc10-5, the anterograde trafficking of Atg9 was affected at the non-permissive temperature (NPT). All these results suggest a role of septins in early stages of autophagy during autophagosome formation.

Due to its central role in growth, proliferation, survival and migration, phosphoinositide 3-kinase (PI3K) is considered as an important drug target in oncology (1). BKM120 is one of the clinically most advanced PI3K inhibitors (PI3Ki), and is currently listed in more than 80 clinical studies aimed at attenuating tumour progression. As an off-target effect, BKM120 was reported to disrupt microtubules (MT) at concentrations around 1 μM (2). Here, we elucidate in detail the structural factors defining PI3K- and tubulin-binding of BKM120, and present a pure PI3K inhibitor (PQR309) and a potent MT disruptor (MTD147) differing from BKM120 by only 1 Dalton. Separation of PI3Ki and MT disruption activities of BKM120 allowed profiling of BKM120 against PQR309 and MTD147: cellular growth profiles of PQR309 clustered with other PI3Ki such as GDC0941/GDC0980, while BKM120 matched MTD147. Both yielded a G2/M cell cycle arrest with typical histone3 phosphorylation. Accumulation of G2/M arrested cells was already evident at concentrations yielding 50% growth inhibition. Interestingly, BKM120 concentrations for 50% cell growth inhibition (with evident G2/M arrest) ranged below or within its reported AUC0-24 levels at day 8 in patient plasma (3,4). This result implies that the two activities of BKM120 cannot be separated, thus complicating the understanding of drug action and impacting on the rational of combination therapies at relevant drug doses. Using X-ray crystallography we found that BKM120 binds to the colchicine pocket on β-tubulin. This study further highlights the importance of the pyrimidine core orientation for tight tubulin binding. Interestingly, activities of regio-isomers of the pyrimidine core are inversed for PI3Ki and tubulin association, and modulate binding by a factor of >30x. Finally, a combination of biochemical, cellular and structural data suggests an inverted orientation of BKM120 in the catalytic cleft of PI3K as previously proposed (6). In summary, the dissection of BKM120 functions allows reassessment of its dominant activity, to increase drug safety, and to flexibly control PI3K and/or MT targeting in combination therapy. 1. M. P. Wymann, R. Schneiter, Nat Rev Mol Cell Biol 9, 162 (2008).; 2. S. M. Brachmann et al., Mol Cancer Ther 11, 1747 (2012). 3. J. C. Bendell et al., J Clin Oncol 30, 282 (2012). 4. C. Saura et al., Clin Cancer Res 20, 1935 (2014). 5. A. E. Prota et al., Science 339, 587 (2013). 6. S. M. Maira et al., Mol Cancer Ther 11, 317 (2012). Citation Format: Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, John E. Burke, Anna Melone, Alison J. Inglis, Ludovico Fusco, Vladimir Cmiljanovic, Natasa Cmiljanovic, Denise Rageot, Katja Bargsten, Gonzalo Saez-Calvo, Olivier Pertz, Amol B. Aher, Anna Akhmanova, Fernando J. Diaz, Doriano Fabbro, Marketa Zvelebil, Roger L. Williams, Michel O. Steinmetz, Matthias P. Wymann. BKM120-mediated G2 arrest: Structural and functional segregation of off-target action and PI3K inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 671. doi:10.1158/1538-7445.AM2015-671