Mild replication stress causes premature centriole disengagement via a sub-critical Plk1 activity under the control of ATR-Chk1.

A tight synchrony between the DNA and centrosome cycle is essential for genomic integrity. Centriole disengagement, which licenses centrosomes for duplication, occurs normally during mitotic exit. We recently demonstrated that mild DNA replication stress typically seen in cancer cells causes premature centriole disengagement in untransformed mitotic human cells, leading to transient multipolar spindles that favour chromosome missegregation. How mild replication stress accelerates the centrosome cycle at the molecular level remained, however, unclear. Using ultrastructure expansion microscopy, we show that mild replication stress induces premature centriole disengagement already in G2 via the ATR-Chk1 axis of the DNA damage repair pathway. This results in a sub-critical Plk1 kinase activity that primes the pericentriolar matrix for Separase-dependent disassembly but is insufficient for rapid mitotic entry, causing premature centriole disengagement in G2. We postulate that the differential requirement of Plk1 activity for the DNA and centrosome cycles explains how mild replication stress disrupts the synchrony between both processes and contributes to genomic instability.

Postbiotic Lipoteichoic acid of probiotic Lactobacillus origin ameliorates inflammation in HT-29 cells and colitis mice.

Lipoteichoic acid (LTA) is a key surface component of probiotic lactobacilli that is involved in important cellular functions including cross talk with the host immune cells. In this study, the anti-inflammatory and ameliorative properties of LTA from probiotic lactobacilli strains were assessed in in vitro HT-29 cells and in vivo colitis mice. The LTA was extracted with n-butanol and its safety was confirmed based on its endotoxin content and cytotoxicity in HT-29 cells. In the Lipopolysaccharide stimulated HT-29 cells, the LTA from the test probiotics evoked a visible but non-significant increase in IL-10 and decrease in TNF-α levels. During the colitis mice study, probiotic LTA treated mice showed substantial improvement in external colitis symptoms, disease activity score and weight gain. The treated mice also showed improvements in key inflammatory markers such as the gut permeability, myeloperoxidase activity and histopathological damages in colon, although non-significant improvements were recorded for the inflammatory cytokines. Furthermore, structural studies by NMR and FTIR revealed increased level of D-alanine substitution in the LTA of LGG strain over MTCC5690. The present study demonstrates the ameliorative effect of LTA as a postbiotic component from probiotics which can be helpful in building effective strategies for combating gut inflammatory disorders.

PLK1 controls centriole distal appendage formation and centrobin removal via independent pathways.

Centrioles are central structural elements of centrosomes and cilia. In human cells, daughter centrioles are assembled adjacent to existing centrioles in S-phase and reach their full functionality with the formation of distal and subdistal appendages one-and-a-half cell cycles later, as they exit their second mitosis. Current models postulate that the centriolar protein centrobin acts as placeholder for distal appendage proteins that must be removed to complete distal appendage formation. Here, we investigated, in non-transformed human epithelial RPE1 cells, the mechanisms controlling centrobin removal and its effect on distal appendage formation. Our data are consistent with a speculative model in which centrobin is removed from older centrioles due to a higher affinity for the newly born daughter centrioles, under the control of the centrosomal kinase PLK1. This removal also depends on the presence of subdistal appendage proteins on the oldest centriole. Removing centrobin, however, is not required for the recruitment of distal appendage proteins, even though this process is equally dependent on PLK1. We conclude that PLK1 kinase regulates centrobin removal and distal appendage formation during centriole maturation via separate pathways.

A CRACker of an adaptor connects dynein-mediated transport to calcium signaling.

Many different adaptor proteins activate the processivity of dynein-dynactin complexes and determine the specific cargo for retrograde transport by binding cargo receptors such as Rab GTP-binding (G) proteins. In this issue, Wang et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201806097) identify two GTPases that can function directly as dynein adaptors during endocytosis and are regulated by calcium.

Incorporating Motility in the Motor: Role of the Hook Protein Family in Regulating Dynein Motility.

Cytoplasmic dynein is a retrograde microtubule-based motor transporting cellular cargo, including organelles, vesicular intermediates, RNA granules, and proteins, thus regulating their subcellular distribution and function. Mammalian dynein associates with dynactin, a multisubunit protein complex that is necessary for the processive motility of dynein along the microtubule tracks. Recent studies have shown that the interaction between dynein and dynactin is enhanced in the presence of a coiled-coil activating adaptor protein, which performs dual functions of recruiting dynein and dynactin to their cargoes and inducing the superprocessive motility of the motor complex. One such family of coiled-coil activating adaptor proteins is the Hook family of proteins that are conserved across evolution with three paralogs in the case of mammals, namely, HOOK1-HOOK3. This Perspective aims to provide an overview of the Hook protein structure and the cellular functions of Hook proteins, with an emphasis on the recent developments in understanding their role as activating dynein adaptors.

The dynein adaptor Hook2 plays essential roles in mitotic progression and cytokinesis.

Hook proteins are evolutionarily conserved dynein adaptors that promote assembly of highly processive dynein-dynactin motor complexes. Mammals express three Hook paralogs, namely Hook1, Hook2, and Hook3, that have distinct subcellular localizations and expectedly, distinct cellular functions. Here we demonstrate that Hook2 binds to and promotes dynein-dynactin assembly specifically during mitosis. During the late G2 phase, Hook2 mediates dynein-dynactin localization at the nuclear envelope (NE), which is required for centrosome anchoring to the NE. Independent of its binding to dynein, Hook2 regulates microtubule nucleation at the centrosome; accordingly, Hook2-depleted cells have reduced astral microtubules and spindle positioning defects. Besides the centrosome, Hook2 localizes to and recruits dynactin and dynein to the central spindle. Dynactin-dependent targeting of centralspindlin complex to the midzone is abrogated upon Hook2 depletion; accordingly, Hook2 depletion results in cytokinesis failure. We find that the zebrafish Hook2 homologue promotes dynein-dynactin association and was essential for zebrafish early development. Together, these results suggest that Hook2 mediates assembly of the dynein-dynactin complex and regulates mitotic progression and cytokinesis.

Multiple Roles, Multiple Adaptors: Dynein During Cell Cycle.

Dynein is an essential protein complex present in most eukaryotes that regulate biological processes ranging from ciliary beating, intracellular transport, to cell division. Elucidating the detailed mechanism of dynein function has been a challenging task owing to its large molecular weight and high complexity of the motor. With the advent of technologies in the last two decades, studies have uncovered a wealth of information about the structural, biochemical, and cell biological roles of this motor protein. Cytoplasmic dynein associates with dynactin through adaptor proteins to mediate retrograde transport of vesicles, mRNA, proteins, and organelles on the microtubule tracts. In a mitotic cell, dynein has multiple localizations, such as at the nuclear envelope, kinetochores, mitotic spindle and spindle poles, and cell cortex. In line with this, dynein regulates multiple events during the cell cycle, such as centrosome separation, nuclear envelope breakdown, spindle assembly checkpoint inactivation, chromosome segregation, and spindle positioning. Here, we provide an overview of dynein structure and function with focus on the roles played by this motor during different stages of the cell cycle. Further, we review in detail the role of dynactin and dynein adaptors that regulate both recruitment and activity of dynein during the cell cycle.

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes.

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit-subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation.