CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex

A conserved γ-tubulin complex–binding domain in CDK5RAP2 stimulates the microtubule-nucleating activity of γ-TuRC

The Spill-Over Impact of the Novel Coronavirus-19 Pandemic on Medical Care and Disease Outcomes in Non-communicable Diseases: A Narrative Review

OBJECTIVES: The coronavirus-19 (COVID-19) pandemic has claimed more than 5 million lives worldwide by November 2021. Implementation of lockdown measures, reallocation of medical resources, compounded by the reluctance to seek help, makes it exceptionally challenging for people with non-communicable diseases (NCD) to manage their diseases. This review evaluates the spill-over impact of the COVID-19 pandemic on people with NCDs including cardiovascular diseases, cancer, diabetes mellitus, chronic respiratory disease, chronic kidney disease, dementia, mental health disorders, and musculoskeletal disorders. METHODS: Literature published in English was identified from PubMed and medRxiv from January 1, 2019 to November 30, 2020. A total of 119 articles were selected from 6,546 publications found. RESULTS: The reduction of in-person care, screening procedures, delays in diagnosis, treatment, and social distancing policies have unanimously led to undesirable impacts on both physical and psychological health of NCD patients. This is projected to contribute to more excess deaths in the future. CONCLUSION: The spill-over impact of COVID-19 on patients with NCD is just beginning to unravel, extra efforts must be taken for planning the resumption of NCD healthcare services post-pandemic

The Eruption of the Candidate Young Star ASASSN-15qi

Outbursts on young stars are usually interpreted as accretion bursts caused by instabilities in the disk or the star-disk connection. However, some protostellar outbursts may not fit into this framework. In this paper, we analyze optical and near-infrared spectra and photometry to characterize the 2015 outburst of the probable young star ASASSN-15qi. The $\sim 3.5$ mag brightening in the $V$ band was sudden, with an unresolved rise time of less than one day. The outburst decayed exponentially by 1 mag for 6 days and then gradually back to the pre-outburst level after 200 days. The outburst is dominated by emission from $\sim10,000$ K gas. An explosive release of energy accelerated matter from the star in all directions, seen in a spectacular cool, spherical wind with a maximum velocity of 1000 km/s. The wind and hot gas both disappeared as the outburst faded and the source the source returned to its quiescent F-star spectrum. Nebulosity near the star brightened with a delay of 10-20 days. Fluorescent excitation of H$_2$ is detected in emission from vibrational levels as high as $v=11$, also with a possible time delay in flux increase. The mid-infrared spectral energy distribution does not indicate the presence of warm dust emission, although the optical photospheric absorption and CO overtone emission could be related to a gaseous disk. Archival photometry reveals a prior outburst in 1976. Although we speculate about possible causes for this outburst, none of the explanations are compelling

Regulation of the microtubule-nucleating activity of the β-tubulin ring complex

Polymerization dynamics enable microtubules to carry out spatial arrangements in response to cellular necessities. Like many other protein polymerization processes, the nucleation steps of microtubule polymerization reactions are slower than the later elongation processes. This allows biological systems to spatially control the reactions. γ-Tubulin complex (γ-TuC) plays a critical role in microtubule nucleation occurring at least at centrosomes, chromatins, and spindle microtubules. However, the control mechanism of γ-TuC-mediated microtubule nucleation remains elusive. CDK5RAP2 is a human microcephaly protein that binds to the γ-TuC and is involved in the centrosomal attachment of γ-tubulin. The γ-TuC-binding domain found in CDK5RAP2 is conserved in Drosophila centrosomin, and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC-tethering proteins in the respective organisms. In the first project, I show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC-mediated nucleation activator (γ-TuNA). The γ-TuNA but not its γ-TuRC-binding deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to the γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2-6. RNAi-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC-mediated microtubule nucleation. I also investigated the novel γ-TuRC-associated protein NME7 and have found that NME7 is a regulatory component of γ-TuRC and plays an important role in γ-TuRC-mediated microtubule nucleation and ciliogenesis. Immunofluorescence studies show that NME7 localizes to centrosomes throughout all stages of cell cycle, in addition to the basal body and axoneme of the early formed cilium. Furthermore, the NME7 centrosomal localization depends on its incorporation into the γ-TuRC. Although NME7 is not required for γ-TuRC assembly or the assembly of the γ-TuRC into centrosomes, RNAi-mediated depletion of NME7 perturbed the centrosomal microtubule nucleation and ciliogenesis. γ-Tubulin is a GDP/GTP binding protein, however, whether GDP/GTP regulates γ-TuRC-mediated microtubule nucleation remains unknown. In the in vitro assays, the microtubule nucleating-activity of γ-TuRC isolated by γ-TuNA was severely compromised by GDP. In the same settings, NME7 stimulated the microtubule nucleating-activity of GDP-preloaded γ-TuRC and promoted GTP binding to γ-tubulin in γ-TuRC regardless of its autophosphorylating activity. Taken together, these results authenticate that NME7 stimulates the γ-TuRC-mediated microtubule nucleation by facilitating the GTP-bound form of γ-TuRC. In summary, γ-TuRC-mediated microtubule nucleation is under multiple controls by γ-TuNA found in CDK5RAP2 and NME7. Multimeric γ-tubulins assembly to γ-TuRC is the prerequisite for the association of γ-TuNA and NME7. The γ-TuNA associates with the γ-TuRC to stimulate γ-TuRC-mediated microtubule nucleation. In addition, NME7 promotes the GTP-bound form of γ-TuRC and facilitates γ-TuRC-mediated microtubule nucleation process

NME7 is a functional component of the gamma-tubulin ring complex

As the primary microtubule nucleator in animal cells, the gamma-tubulin ring complex (gamma TuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the gamma TuRC is regulated. Recently, isolated gamma TuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a gamma TuRC component that regulates the microtubule-nucleating activity of the gamma TuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the gamma TuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the gamma TuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect gamma TuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes gamma TuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the gamma TuRC in a kinase-dependent manner to facilitate microtubule nucleation

Dynamic Recruitment of CDK5RAP2 to Centrosomes Requires Its Association with Dynein

CDK5RAP2 is a centrosomal protein known to be involved in the regulation of the gamma-tubulin ring complex and thus the organization of microtubule arrays. However, the mechanism by which CDK5RAP2 is itself recruited to centrosomes is poorly understood. We report here that CDK5RAP2 displays highly dynamic attachment to centrosomes in a microtubule-dependent manner. CDK5RAP2 associates with the retrograde transporter dynein-dynactin and contains a sequence motif that binds to dynein light chain 8. Significantly, disruption of cellular dynein-dynactin function reduces the centrosomal level of CDK5RAP2. These results reveal a key role of the dynein-dynactin complex in the dynamic recruitment of CDK5RAP2 to centrosomes

CDK5RAP2 associates with dynein.

<p>(A) Extracts of HEK293T cells expressing FLAG-CDK5RAP2 were incubated with taxol-polymerized microtubules in the absence or presence of AMP-PNP; control samples lacked polymerized microtubules. After incubation, microtubules were spun down through a sucrose cushion and the pellets were immunoblotted with an anti-FLAG antibody for CDK5RAP2; α-tubulin in these pellets was quantified by staining with anti-α-tubulin antibody (n = 3, <i>p</i><0.001). (B) DIC was immunoprecipitated from HEK293T extracts using an anti-DIC antibody, normal mouse IgG served as a control. The immunoprecipitates (“IP” here and in other figures) and cell extracts were probed for DIC and CDK5RAP2 by Western blotting (“WB” here and in other figures). (C) Mapping the DIC-binding region in CDK5RAP2. Anti-DIC immunoprecipitation was performed on HEK293T ectopically expressing CDK5RAP2 fragments. Immunoprecipitates and cell extracts were analyzed for DIC and for the CDK5RAP2 fragments with anti-FLAG. A control immunoprecipitation (Ctrl) was performed using normal mouse IgG on lysates of 706–1893-expressing cells.</p

The dynamic recruitment of CDK5RAP2 to centrosomes depends on microtubules and dynein.

<p>(A) Immunoblotting of extracts prepared from MDA-MB-231 cells stably expressing GFP-CDK5RAP2. Cells were lysed and extracts were stained with an anti-CDK5RAP2 antibody to detect GFP-CDK5RAP2 and endogenous CDK5RAP2 in either the stable cell line or the parent cell line. Anti-α-tubulin was used as internal control. (B) GFP-CDK5RAP2 is localized on centrosomes. Cells transfected with GFP-CDK5RAP2 were stained with anti-γ-tubulin to label centrosomes. (C) Centrosomal recruitment of CDK5RAP2. FRAP experiments were performed on cells expressing GFP-CDK5RAP2 that were treated with or without nocodazole (noco, nocodazole 10 μM for 1 h) before starting the assays. The intensity of the CDK5RAP2 signal relative to its original level (before bleaching) on centrosomes (arrowheads) is plotted on the right; the intensity recovered to 60% of the original intensity (n = 5). (D) Effect of the disruption of dynein-dynactin on the centrosomal accumulation of CDK5RAP2. The centrosomal level of CDK5RAP2 was determined in cells un-transfected and transfected with mCherry-CC1; relative amounts of centrosomal CDK5RAP2 are shown on the right (n≥30 cells for each case; error bars, S.D. <i>p</i><0.001). Arrows and arrowheads point to centrosomes in CC1-positive and CC1-negative cells. Centrin serves as an indicator of centrosomes. Centrosomes are magnified in insets. Scale bar, 10 μm. Representative results of at least three separate experiments are shown here.</p