Multimerization of a disordered kinetochore protein promotes accurate chromosome segregation by localizing a core dynein module.

Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation by a poorly understood pathway. Here, we identify a linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ contribute to the linkage. Furthermore, a minimal segment of Spc105 with a propensity to multimerize and which contains protein binding motifs is sufficient to link Spc105 to RZZ/dynein. Deletion of the minimal region from Spc105 compromises the recruitment of its binding partners to kinetochores and elevates chromosome missegregation due to merotelic attachments. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 contributes to localizing a core pool of RZZ that promotes accurate chromosome segregation.

Human dynein-dynactin is a fast processive motor in living cells.

Minus-end directed transport along microtubules in eukaryotes is primarily mediated by cytoplasmic dynein and its cofactor dynactin. Significant advances have been made in recent years characterizing human dynein-dynactin structure and function using in vitro assays, however, there is limited knowledge about the motile properties and functional organization of dynein-dynactin in living human cells. Total internal reflection fluorescence microscopy (TIRFM) of CRISPR-engineered human cells is employed here to visualize fluorescently tagged dynein heavy chain (DHC) and p50 with high spatio-temporal resolution. We find that p50 and DHC exhibit indistinguishable motility properties in their velocities, run lengths, and run times. The dynein-dynactin complexes are fast (∼1.2 µm/s) and typically run for several microns (∼2.7 µm). Quantification of the fluorescence intensities of motile puncta reveals that dynein-dynactin runs are mediated by at least one DHC dimer while the velocity is consistent with that measured for double dynein (two DHC dimers) complexes in vitro.

An intrinsically disordered kinetochore protein coordinates mechanical regulation of chromosome segregation by dynein.

Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation through a poorly understood pathway. Here we identify a physical linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ mediate the connection of Spc105 to dynein. Furthermore, a minimal segment of Spc105 that contains regions with a propensity to multimerize and binding motifs for Bub1 and BubR1 is sufficient to functionally link Spc105 to RZZ and dynein. Deletion of the minimal region from Spc105 reduces recruitment of its binding partners to bioriented kinetochores and causes chromosome mis-segregation. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and higher-order oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 is required to recruit a core pool of RZZ that modulates microtubule attachment stability to promote accurate chromosome segregation.

Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is continuously evolving, and this poses a major threat to antibody therapies and currently authorized Coronavirus Disease 2019 (COVID-19) vaccines. It is therefore of utmost importance to investigate and predict the putative mutations on the spike protein that confer immune evasion. Antibodies are key components of the human immune system's response to SARS-CoV-2, and the spike protein is a prime target of neutralizing antibodies (nAbs) as it plays critical roles in host cell recognition, fusion, and virus entry. The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations. In this review, we discuss (1) the emerging SARS-CoV-2 variants; (2) the structural basis for antibody-mediated neutralization of SARS-CoV-2 and nAb classification; and (3) identification of the RBD escape mutations for several antibodies that resist antibody binding and neutralization. These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines.

A celebration of the 25th anniversary of chromatin-mediated spindle assembly.

Formation of a bipolar spindle is required for the faithful segregation of chromosomes during cell division. Twenty-five years ago, a transformative insight into how bipolarity is achieved was provided by Rebecca Heald, Eric Karsenti, and colleagues in their landmark publication characterizing a chromatin-mediated spindle assembly pathway in which centrosomes and kinetochores were dispensable. The discovery revealed that bipolar spindle assembly is a self-organizing process where microtubules, which possess an intrinsic polarity, polymerize around chromatin and become sorted by mitotic motors into a bipolar structure. On the 25th anniversary of this seminal paper, we discuss what was known before, what we have learned since, and what may lie ahead in understanding the bipolar spindle.

Global lockdown: An effective safeguard in responding to the threat of COVID-19.

RATIONALE, AIMS, AND OBJECTIVES: The recent outbreak of coronavirus (COVID-19) has infected around 1 560 000 individuals till 10 April 2020, which has resulted in 95 000 deaths globally. While no vaccine or anti-viral drugs for COVID-19 are available, lockdown acts as a protective public health measures to reduce human interaction and lower transmission. The study aims to explore the impact of delayed planning or lack of planning for the lockdown and inadequate implementation of the lockdown, on the transmission rate of COVID-19. METHOD: Epidemiological data on the incidence and mortality of COVID-19 cases as reported by public health authorities were accessed from six countries based on total number of infected cases, namely, United States and Italy (more than 100 000 cases); United Kingdom, and France (50 000-100 000 cases), and India and Russia (6000-10 000 cases). The Bayesian inferential technique was used to observe the changes (three points) in pattern of number of cases on different duration of exposure (in days) in these selected countries 1 month after World Health Organization (WHO) declaration about COVID-19 as a global pandemic. RESULTS: On comparing the pattern of transmission rates observed in these six countries at posterior estimated change points, it is found that partial implementation of lockdown (in the United States), delayed planning in lockdown (Russia, United Kingdom, and France), and inadequate implementation of the lockdown (in India and Italy) were responsible to the spread of infections. CONCLUSIONS: In order to control the spreading of COVID-19, like other national and international laws, lockdown must be implemented and enforced. It is suggested that on-time or adequate implementation of lockdown is a step towards social distancing and to control the spread of this pandemic.

COVID-19 pandemic: Insights into structure, function, and hACE2 receptor recognition by SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a newly emerging, highly transmissible, and pathogenic coronavirus in humans that has caused global public health emergencies and economic crises. To date, millions of infections and thousands of deaths have been reported worldwide, and the numbers continue to rise. Currently, there is no specific drug or vaccine against this deadly virus; therefore, there is a pressing need to understand the mechanism(s) through which this virus enters the host cell. Viral entry into the host cell is a multistep process in which SARS-CoV-2 utilizes the receptor-binding domain (RBD) of the spike (S) glycoprotein to recognize angiotensin-converting enzyme 2 (ACE2) receptors on the human cells; this initiates host-cell entry by promoting viral-host cell membrane fusion through large-scale conformational changes in the S protein. Receptor recognition and fusion are critical and essential steps of viral infections and are key determinants of the viral host range and cross-species transmission. In this review, we summarize the current knowledge on the origin and evolution of SARS-CoV-2 and the roles of key viral factors. We discuss the structure of RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and its significance in drug discovery and explain the receptor recognition mechanisms of coronaviruses. Further, we provide a comparative analysis of the SARS-CoV and SARS-CoV-2 S proteins and their receptor-binding specificity and discuss the differences in their antigenicity based on biophysical and structural characteristics.

Delineating the contribution of Spc105-bound PP1 to spindle checkpoint silencing and kinetochore microtubule attachment regulation.

Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore-microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore-microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

Direct observation of branching MT nucleation in living animal cells.

Centrosome-mediated microtubule (MT) nucleation has been well characterized; however, numerous noncentrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multicolor TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells during anaphase. The steps of a stereotypical branching nucleation event entailed augmin binding to a mother MT and recruitment of γ-TuRC after 15 s, followed by nucleation 16 s later of a daughter MT at a 36° branch angle. Daughters typically remained attached throughout their ∼40-s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require Drosophila TPX2, enhanced localized RhoA activation during cytokinesis.

Classical and Emerging Regulatory Mechanisms of Cytokinesis in Animal Cells.

The primary goal of cytokinesis is to produce two daughter cells, each having a full set of chromosomes. To achieve this, cells assemble a dynamic structure between segregated sister chromatids called the contractile ring, which is made up of filamentous actin, myosin-II, and other regulatory proteins. Constriction of the actomyosin ring generates a cleavage furrow that divides the cytoplasm to produce two daughter cells. Decades of research have identified key regulators and underlying molecular mechanisms; however, many fundamental questions remain unanswered and are still being actively investigated. This review summarizes the key findings, computational modeling, and recent advances in understanding of the molecular mechanisms that control the formation of the cleavage furrow and cytokinesis.

Microtubule plus-ends act as physical signaling hubs to activate RhoA during cytokinesis.

Microtubules (MTs) are essential for cleavage furrow positioning during cytokinesis, but the mechanisms by which MT-derived signals spatially define regions of cortical contractility are unresolved. In this study cytokinesis regulators visualized in Drosophila melanogaster (Dm) cells were found to localize to and track MT plus-ends during cytokinesis. The RhoA GEF Pebble (Dm ECT2) did not evidently tip-track, but rather localized rapidly to cortical sites contacted by MT plus-tips, resulting in RhoA activation and enrichment of myosin-regulatory light chain. The MT plus-end localization of centralspindlin was compromised following EB1 depletion, which resulted in a higher incidence of cytokinesis failure. Centralspindlin plus-tip localization depended on the C-terminus and a putative EB1-interaction motif (hxxPTxh) in RacGAP50C. We propose that MT plus-end-associated centralspindlin recruits a cortical pool of Dm ECT2 upon physical contact to activate RhoA and to trigger localized contractility.

NOD is a plus end-directed motor that binds EB1 via a new microtubule tip localization sequence.

Chromosome congression, the process of positioning chromosomes in the midspindle, promotes the stable transmission of the genome to daughter cells during cell division. Congression is typically facilitated by DNA-associated, microtubule (MT) plus end-directed motors called chromokinesins. The Drosophila melanogaster chromokinesin NOD contributes to congression, but the means by which it does so are unknown in large part because NOD has been classified as a nonmotile, orphan kinesin. It has been postulated that NOD promotes congression, not by conventional plus end-directed motility, but by harnessing polymerization forces by end-tracking on growing MT plus ends via a mechanism that is also uncertain. Here, for the first time, it is demonstrated that NOD possesses MT plus end-directed motility. Furthermore, NOD directly binds EB1 through unconventional EB1-interaction motifs that are similar to a newly characterized MT tip localization sequence. We propose NOD produces congression forces by MT plus end-directed motility and tip-tracking on polymerizing MT plus ends via association with EB1.

A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA.

Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

Cohesin rings devoid of Scc3 and Pds5 maintain their stable association with the DNA.

Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.

Equilibrium and kinetics of the unfolding and refolding of Escherichia coli Malate Synthase G monitored by circular dichroism and fluorescence spectroscopy.

The equilibrium and kinetics studies of an 82 kDa large monomeric Escherichia coli protein Malate Synthase G (MSG) was investigated by far and near-UV CD, intrinsic tryptophan fluorescence and extrinsic fluorescence spectroscopy. We find that despite of its large size, folding is reversible, in vitro. Equilibrium unfolding process of MSG exhibited three-state transition thus, indicating the presence of at least a stable equilibrium intermediate. Thermodynamic parameters suggest this intermediate resembles the unfolded state. However, the equilibrium intermediate exhibits pronounced secondary structure as measured by far-UV CD, partial tertiary structure as delineated by near-UV CD, compactness (m value) and exposed hydrophobic surface area as assessed by ANS binding, typically depicting a molten globule state. The stopped-flow kinetic data provide clear evidence for the presence of a burst phase during the refolding pathway due to the formation of an early Intermediate, within the dead time of the instrument. Refolding from 4 M to various lower concentrations until 0.4 M of GdnHCl follow biphasic kinetics at lower concentrations of GdnHCl (<0.8 M), whereas monophasic kinetics at concentrations above 1.5 M. Also, rollover in the refolding and unfolding limbs of chevron plot verifies the presence of a fast kinetic intermediate at lower concentration of GdnHCl. Based upon the above observations we hereby propose the folding pathway of a large multi-domain protein Malate Synthase G.

GroEL assisted folding of large polypeptide substrates in Escherichia coli: Present scenario and assignments for the future.

Escherichia coli chaperonins GroEL and GroES are indispensable for survival and growth of the cell since they provide essential assistance to the folding of many newly translated proteins in the cell. Recent studies indicate that a substantial portion of the proteins involved in the host pathways are completely dependent on GroEL-GroES for their folding and hence providing some explanation for why GroEL is essential for cell growth. Many proteins either small-single domain or large multidomains require assistance from GroEL-ES during their lifetime. Proteins of size up to approximately 70kDa can fold via the cis mechanism during GroEL-ES assisted pathway, but other proteins (>70kDa) that cannot be pushed inside the cavity of GroEL-ATP complex upon binding of GroES fold by an evolved mechanism called trans. In recent years, much work has been done on revealing facts about the cis mechanism involving the GroEL assisted folding of small proteins whereas the trans mechanism with larger polypeptide substrates still remains under cover. In order to disentangle the role of chaperonin GroEL-GroES in the folding of large E. coli proteins, this review discusses a number of issues like the range of large polypeptide substrates acted on by GroEL. Do all these substrates need the complete chaperonin system along with ATP for their folding? Does GroEL act as foldase or holdase during the process? We conclude with a discussion of the various queries that need to be resolved in the future for an extensive understanding of the mechanism of GroEL mediated folding of large substrate proteins in E. coli cytosol.

A new biologically active flavonol glycoside from Psoralea corylifolia (Linn.).

A new biologically active flavonol glycoside (1) mp 264-265 degrees C, C32H38O20, [M]+ 742 (EIMS) has been isolated from the methanol-soluble fraction of the defatted seeds of Psoralea corylifolia (Linn.). It was characterised as the new flavonol glycoside 3,5,3',4'-tetrahydroxy-7-methoxyflavone-3'-O-alpha-L-xylopyranosyl(1-->3)-O-alpha-L-arabinopyranosyl(1-->4)-O-beta-D-galactopyranoside by several colour reactions, spectral analysis and chemical degradations. Compound 1 showed anti-microbial activity against various bacteria and fungi.

A new biologically active flavone glycoside from the seeds of Cassia fistula (Linn.).

A new bioactive flavone glycoside 1 [mp 252-254 degrees C, C28H32O16, [M]+ 624 (EIMS)] was isolated from the acetone soluble fraction of the defatted seeds of Cassia fistula (Linn.). It was characterized as a new bioactive flavone glycoside 5,3',4'-tri-hydroxy-6-methoxy-7-O-alpha-L-rhamnopyranosyl-(1 --> 2)-O-beta-D-galactopyranoside by several colour reactions, spectral analysis and chemical degradations. Compound 1 showed anti-microbial activity.