Biology subjects

Urbach, S.

Publications and source records attributed to Urbach, S..

23 records · Page 2Linked to original sources

bioRxiv2021

The beta-tubulin isotype TUBB6 controls microtubule and actin dynamics in osteoclasts.

Osteoclasts are bone resorbing cells that participate in the maintenance of bone health. Pathological increase in osteoclast activity causes bone loss eventually resulting in osteoporosis. Actin cytoskeleton of osteoclasts organizes into a belt of podosomes, which sustains the bone resorption apparatus and is maintained by microtubules. Better understanding of the molecular mechanisms regulating osteoclast cytoskeleton is key to understand the mechanisms of bone resorption, in particular to propose new strategies against osteoporosis. We reported recently that {beta}-tubulin isotype TUBB6 is key for cytoskeleton organization in osteoclasts and for bone resorption. Here, using an osteoclast model CRISPR/Cas9 KO for Tubb6, we show that TUBB6 controls both microtubule and actin dynamics in osteoclasts. Osteoclasts KO for Tubb6 have reduced microtubule growth speed with longer growth life time, higher levels of acetylation and smaller EB1-caps. On the other hand, lack of TUBB6 increases podosome life time while the belt of podosomes is destabilized. Finally, we performed proteomic analyses of osteoclast microtubule-associated protein enriched fractions. This highlighted ARHGAP10 as a new microtubule-associated protein, which binding to microtubules appears to be negatively regulated by TUBB6. ARHGAP10 is a negative regulator of CDC42 activity, which participates in actin organization in osteoclasts. Our results suggest that TUBB6 plays a key role in the control of microtubule and actin cytoskeleton dynamics in osteoclasts. Moreover, by controlling ARHGAP10 association with microtubules, TUBB6 may participate in the local control CDC42 activity to ensure efficient bone resorption.

cell biology↗

bioRxiv2020

TopBP1 assembles nuclear condensates to switch on ATR signalling.

ATR checkpoint signalling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micron-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse and co-localise with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slowdown replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR-activation domain disrupt TopBP1 condensation and, consequently, ATR/Chk1 signalling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signalling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

cell biology↗

bioRxiv2019

Quantitative proteomics and transcriptomics exploration of primary myeloid cells: importance of β tubulin isotypes for bone resorption.

Among hematopoietic cells. osteoclasts (Oc) and immature dendritic cells (Dc) are closely related myeloid cells with distinct functions; Oc participate skeleton maintenance while Dc sample the environment for foreign antigens. Such specificities rely on profound modifications of gene and protein expression during Oc and Dc differentiation. We provide global proteomic and transcriptomic analyses of primary mouse Oc and Dc. based on original SILAC and RNAseq data. We established specific signatures for Oc and Dc including genes and proteins of unknown functions. In particular. we showed that Oc and Dc have the same and {beta} tubulin isotypes repertoire but that Oc express much more {beta} tubulin isotype Tubb6. In both mouse and human Oc. we demonstrate that elevated expression of Tubb6 in Oc is necessary for correct podosomes organization and thus for the structure of the sealing zone. which sustains the bone resorption apparatus. Hence. lowering Tubb6 expression hindered Oc resorption activity. Overall. we highlight here potential new regulators of Oc and Dc biology and illustrate the functional importance of the tubulin isotype repertoire in the biology of differentiated cells. Summary statementThis study provides original proteomic and transcriptomic data of primary myeloid cells. The analysis led to signatures for osteoclasts and for immature dendritic cells including potential new regulators of their specific biology. RNA interference showed in particular that {beta} tubulin isotype Tubb6 participates in osteoclast podosome patterning. sealing zone structure and in the resorption activity.

cell biology↗

bioRxiv2019

Inheritance of Chromatin Proteins in Budding Yeast: metabolic gene regulators TUP1, FPR4 and Rpd3L are retained in the mother cell.

Asymmetric division is a prerequisite for cellular differentiation. Phenotypic transformation during differentiation is a poorly understood epigenetic phenomenon, in which chromatin theoretically plays a role. The assumption that chromatin components segregate asymmetrically in asymmetric divisions has however not been systematically tested. We have developed a live cell imaging method to measure how 18 chromatin proteins are inherited in asymmetric divisions of budding yeast. We show that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3, Fpr4 and Tup1, which are retained in the mother. Mother retention seems to be the norm for low abundance proteins with the exception of Sir2 and the linker histone H1. Our in vivo analysis of chromatin protein behavior in single cells highlights general trends in protein biology during the cell cycle such as coupled protein synthesis and decay, and a correlation between half-lives and cell cycle duration.

cell biology↗

bioRxiv2017

Initiation of DNA replication requires actin dynamics and formin activity

Nuclear actin influences transcription in a manner dependent on its dynamics of polymerisation and nucleocytoplasmic translocation. Using human somatic cells and transcriptionally-silent Xenopus egg extracts, we show that actin dynamics is also required for DNA replication. We identify many actin regulators in replicating nuclei from Xenopus egg extracts, and show that in human cells, nuclear actin filaments form in early G1 and disassemble prior to S-phase. In either system, treatments that stabilise nuclear actin filaments abrogate nuclear transport and initiation of DNA replication. Mechanistically, actin directly binds RanGTP-importin complexes and disruption of its dynamics hinders cargo release. This prevents both nuclear pore complex (NPC) formation and active nuclear transport, which we show is required throughout DNA replication. Nuclear formin activity is required for two further steps: loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin and initiation of DNA replication. Thus, actin dynamics and formins are involved in several nuclear processes essential for cell proliferation.

cell biology↗