Biology subjects

Desai, A.

Publications and source records attributed to Desai, A..

6 recordsLinked to original sources

bioRxiv2018

Ancestral Roles of the Fam20C Family of Secreted Protein Kinases Revealed by Functional Analysis in C. elegans

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the C. elegans Fam20C ortholog, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains inter-cellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in this context. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.

cell biology↗

bioRxiv2018

Generation of functional human adipose tissue in mice from primed progenitor cells

AbstarctAdipose tissue is used extensively in reconstructive and regenerative therapies, but transplanted fat often undergoes inflammation and cell death, requiring further revision surgery. We report that functional human adipose tissue can be generated from mesenchymal progenitor cells in-vivo, providing an alternative approach to its therapeutic use. We leveraged previous findings that progenitor cells within the vasculature of human adipose tissue robustly proliferate in 3-dimensional culture under proangiogenic conditions. Implantation of these progenitor cells into immunocompromised mice results in differentiation towards non-adipocyte fates, incapable of generating a distinct tissue structure. However, priming of these progenitor cells in-vitro towards adipogenic differentiation results in formation of functional adipose tissue in-vivo. Mechanistically, priming induces the expression of genes encoding specific extracellular matrix and remodeling proteins, and induces extensive vascularization by host blood vessels. In comparison, grafts from adipose tissue obtained by liposuction undergo poor vascularization, adipocyte death, cyst formation, calcification and inefficient adiponectin secretion. Thus, primed mesenchymal adipose tissue progenitors reveal mechanisms of human adipose tissue development, and have potential to improve outcomes in reconstructive and regenerative medicine.

bioengineering↗

bioRxiv2017

CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Formation of gametes ultimately requires bridge closure. Here, we investigate the contribution of the conserved bridge component centralspindlin to oocyte production in C. elegans. Centralspindlin is composed of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6, which are both essential for cytokinesis. In contrast, we show that oocyte production by the syncytial germline requires CYK-4 but not ZEN-4. Longitudinal imaging after conditional CYK-4 inactivation revealed a role in oocyte cellularization, rather than in generation of syncytial compartments. CYK-4s lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were individually important for oocyte cellularization and for targeting CYK-4 to bridges, where it contributes to enrichment of active RhoA. These results identify a C1-GAP module in CYK-4 that recruits it to bridges in the germline and directs their closure to produce oocytes.\n\nIMPACT STATEMENTThe CYK-4 subunit of centralspindlin, a broadly conserved component of intercellular bridges across metazoa, is required for the cytokinesis-like closure of intercellular bridges that cellularizes oocytes to separate them from germline syncytia.\n\nMAJOR SUBJECT AREASCell Biology, Developmental Biology & Stem Cells

cell biology↗

bioRxiv2017

Positive Feedback Between Contractile Ring Myosin and Ring-Directed Cortical Flow Drives Cytokinesis

During cytokinesis, an equatorial actomyosin contractile ring constricts at a relatively constant overall rate despite its progressively decreasing size. Thus, the per-unit-length rate of ring closure increases as ring perimeter decreases. To understand this acceleration, we monitored cortical surface and ring component dynamics during the first division of the C. elegans embryo. We show that the polar cortex expands during ring constriction to provide the cortical surface area required for division. Polar expansion also allows ring myosin to compress cortical surface along the pole-to-pole axis, leading to a continuous flow of cortical surface into the ring. We propose that feedback between ring myosin and compression-driven cortical flow drives an exponential increase in the amount of ring myosin that maintains the high overall closure rate as ring perimeter decreases. We further show that an analytical mathematical formulation of the proposed feedback, called the Compression Feedback model, recapitulates the experimental observations.\n\nIMPACT STATEMENTDuring cytokinesis, positive feedback between myosin motors in the contractile ring and compression-driven cortical flow along the axis perpendicular to the ring drives constriction rate acceleration to ensure timely cell separation.\n\nMAJOR SUBJECT AREASCell biology, Computational and Systems Biology

cell biology↗

bioRxiv2017

Preservation of Chromatin Organization after Acute Loss of CTCF in Mouse Embryonic Stem Cells

The CCCTC-binding factor (CTCF) is widely regarded as a key player in chromosome organization in mammalian cells, yet direct assessment of the impact of loss of CTCF on genome architecture has been difficult due to its essential role in cell proliferation and early embryogenesis. Here, using auxin-inducible degron techniques to acutely deplete CTCF in mouse embryonic stem cells, we show that cell growth is severely slowed yet chromatin organization remains largely intact after loss of CTCF. Depletion of CTCF reduces interactions between chromatin loop anchors, diminishes occupancy of cohesin complex genome-wide, and slightly weakens topologically associating domain (TAD) structure, but the active and inactive chromatin compartments are maintained and the vast majority of TAD boundaries persist. Furthermore, transcriptional regulation and histone marks associated with enhancers are broadly unchanged upon CTCF depletion. Our results suggest CTCF-independent mechanisms in maintenance of chromatin organization.

genomics↗

bioRxiv2017

A toolkit for tissue-specific protein degradation in C. elegans

Proteins essential for embryo production, cell division, and early embryonic events are frequently re-utilized later in embryogenesis, during organismal development, or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that utilizes expression of a fusion between a GFP-targeting nanobody and SOCS-box containing ubiquitin ligase adaptor to target GFP tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues--the epidermis, intestine, body wall muscle, sensory neurons, and touch neurons--where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.

developmental biology↗