As the main microtubule-organizing center in animal cells, the centrosome has a fundamental role in cell function. Surrounding the centrioles, the Pericentriolar material (PCM) provides a dynamic platform for nucleating microtubules. While the PCM’s importance is established, its amorphous electron-dense nature has made it refractory to structural investigation. By using SIM and STORM sub-diffraction resolution microscopies to visualize proteins critical for centrosome maturation, we demonstrate that the PCM is organized into two major structural domains: a layer juxtaposed to the centriole wall, and proteins extending further away from the centriole organized in a matrix. Analysis of Pericentrin-like protein (Plp) reveals that its C-terminus is positioned at the centriole wall, it radiates outward into the matrix and is organized in clusters having quasi-nine-fold symmetry. By RNAi we show that Plp fibrils are required for interphase recruitment and proper mitotic assembly of the PCM matrix.

Super-resolution microscopy (SRM) methods have allowed scientists to exceed the diffraction limit of light, enabling the discovery and investigation of cellular structures at the nanometer scale, from individual proteins to entire organelles. In this review we survey the application of SRM in elucidating the structure of macromolecules in the native cellular environment. We emphasize how SRM can generate molecular maps of protein complexes and extract quantitative information on the number, size, distribution, and spatial organization of macromolecules. We discuss both the novel information that can be generated through SRM as well as the experimental considerations to examine while conducting such studies. With the increasing popularity of SRM in the biological sciences, this review will serve as a tool to navigate the range of applications and harness the power of SRM to elucidate biological structures.

Centrosomes are conserved organelles that are essential for accurate cell division and cilium formation. A centrosome consists of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM) that is essential for the centrosome’s function. In this study, we show that Sas-4 provides a scaffold for cytoplasmic complexes (named S-CAP), which include CNN, Asl and D-PLP, proteins that are all found in the centrosomes at the vicinity of the centriole. When Sas-4 is absent, nascent procentrioles are unstable and lack PCM, and functional centrosomes are not generated. When Sas-4 is mutated, so that it cannot form S-CAP complexes, centrosomes are present but with dramatically reduced levels of PCM. Finally, purified S-CAP complexes or recombinant Sas-4 can bind centrosomes stripped of PCM, whereas recombinant CNN or Asl cannot. In summary, PCM assembly begins in the cytosol where Sas-4 provides a scaffold for pre-assembled cytoplasmic complexes before tethering of the complexes in a centrosome.

The inheritance of the centrosome during human fertilization remains mysterious. Here we show that the sperm centrosome contains, in addition to the known typical barrel-shaped centriole (the proximal centriole, PC), a surrounding matrix (pericentriolar material, PCM), and an atypical centriole (distal centriole, DC) composed of splayed microtubules surrounding previously undescribed rods of centriole luminal proteins. The sperm centrosome is remodeled by both reduction and enrichment of specific proteins and the formation of these rods during spermatogenesis. In vivo and in vitro investigations show that the flagellum-attached, atypical DC is capable of recruiting PCM, forming a daughter centriole, and localizing to the spindle pole during mitosis. Altogether, we show that the DC is compositionally and structurally remodeled into an atypical centriole, which functions as the zygote’s second centriole. These findings now provide novel avenues for diagnostics and therapeutic strategies for male infertility, and insights into early embryo developmental defects.

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.

The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The pericentriolar material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules, and acts as a nexus for molecular signaling. While great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance under the electron microscope have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supra-molecular organization of organelles in cells.

ith more than 53 million confirmed cases of COVID-19 and 1.3 million associated deaths worldwide (World Health Organization, 13 November 2020), there is an urgent need to understand the molecular mechanism of infection and disease to identify patients' susceptibilities and targets for therapeutic intervention. ACE2 is the main viral entry point for coronavirus N63, SARS-CoV and SARS-CoV-2, which cause severe acute respiratory syndromes, the last being responsible for COVID-19 in humans 1-4. ACE2 binds to the S1 domain of trimeric SARS-CoV spike (S) glycoprotein 1 and SARS-CoV-2 S protein 5 , which is primed by TMPRSS2 (ref. 6). Cellular entry of SARS-CoV is dependent on the extracellular domain of ACE2 being cleaved by TMPRSS2 protease at Arg 697 and Lys 716, and the transmembrane domain of ACE2 internalized with the virus 7-9. ACE2 is a carboxypeptidase with several known physiological functions including regulation of blood pressure, salt and water balance in mammals 10,11 , amino acid uptake in the small intestine 12,13 , and glucose homeostasis and pancreatic β-cell function 14,15. Interestingly, ACE2 has been suggested to play an important role in protection from acute lung injury 16-19. ACE2 expression in different tissues is controlled by multiple promoter elements 20-22. In human nasal epithelia and lung tissue, ACE2 expression has been reported to be interferon (IFN) regulated, with evidence of STAT1-, STAT3-, IRF8-and IRF1-binding sites within the ACE2 promoter 23. Activation of IFN-responsive genes is an important antiviral defense pathway in humans, and both interferon and influenza exposure have been reported to increase ACE2 expression in the human airway 23. Bulk and single-cell RNA-sequencing (scRNA-seq) data 24 detect low-level expression of ACE2 in multiple tissues 25. ACE2 expression in the airways is relatively high in nasal epithelium and progressively lower in the bronchial and alveolar regions; this expression profile correlates with levels of infection of SARS-CoV-2 isolates from patients in different airway compartments 26. Consistently, SARS-CoV-2 viral loads have been found to be higher in swabs taken from the nose than swabs taken from the throat of COVID-19 patients 27. Highest ACE2 expression is seen in goblet and ciliated cells of the nasal epithelium 25 , and ACE2 protein localizes to the membrane of motile cilia of respiratory tract epithelia 28. Consistent with this, SARS-CoV-2 has been detected in situ in ciliated airway cells and upper airway epithelium, in addition to pulmonary A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection

Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.