Biology subjects

Desai, A.

Publications and source records attributed to Desai, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis

Many proteins required for embryogenesis are expressed in multiple tissues and across developmental time, often with tissue- and stage-specific roles. To resolve where and when proteins function, we developed a tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis. We generated a plasmid set compatible with single-copy insertion or high-copy extrachromosomal array generation that employs tissue-specific promoters (active in the germline, intestine, muscle, pharynx, neuroblasts, neurons, sensory neurons, epidermis, and pan soma) to drive an operon expressing an optimized auxin-dependent plant F-box factor TIR1 and, in most cases, a fluorescent histone. Using a ubiquitous nuclear envelope marker as a test substrate, we identified single-copy insertions and arrays that exhibit efficient auxin-dependent, tissue-specific target degradation. We also establish a workflow that ensures all functionally important isoforms of a target are tagged and that degradation reproduces the loss-of-function phenotype prior to tissue-specific analysis. Applying the toolkit to the broadly expressed Rho kinase LET-502, we find that degradation in the epidermis, but not in other embryonic tissues, disrupts elongation. Unexpectedly, temporal analysis revealed that LET-502 is required for late as well as early elongation.

developmental biology↗

bioRxiv2026

Inhibitor-2 directs formation of PP1 holoenzymes through a docking motif-dependent transfer of catalytic subunits to adapters

The catalytic subunits of protein phosphatase 1 (PP1) achieve spatiotemporal substrate specificity by assembling with diverse regulatory adapters to form holoenzymes. Three conserved proteins--Sds22, Inhibitor-2 and Inhibitor-3--facilitate loading of PP1 catalytic subunits (PP1cs) onto adapters. We show here that Inhibitor-2 is central to a dynamic cycle that directs formation of adapter-bound PP1 holoenzymes. Inhibitor-2 engages PP1cs via two adapter-like docking motifs (RVxF and SILK) and an active site-binding inhibitory region. While Inhibitor-2 depletion produced moderate phenotypes, mutation of its RVxF docking motif caused severe defects resembling global PP1c inhibition. The RVxF mutant did not prevent PP1c binding or reduce PP1c stability but inhibited formation of adapter-bound holoenzymes. The severe effects of the RVxF mutation were suppressed by linked mutation of the inhibitory active site-binding motif. These results suggest that Inhibitor-2 is integral to a dynamic cycle that delivers PP1cs to adapters, with its RVxF motif being critical for coupling relief of active site inhibition to adapter handoff.

cell biology↗

bioRxiv2025

The mitotic stopwatch synergizes with mild p53 activation to halt cell proliferation

The mitotic stopwatch suppresses proliferation of cell lineages experiencing prolonged mitosis that are prone to chromosome missegregation and tumorigenesis. It converts extended mitotic duration into heritable USP28-53BP1 complexes that stabilize p53 and accumulate over generations. To identify genes whose knockout activates the stopwatch, we performed a CRISPR/Cas9 screen comparing dropout kinetics of essential-gene gRNAs in cells lacking versus possessing the stopwatch. Two classes of knockouts emerged: one (27/60 top hits) that prolonged mitosis, and another (33/60 top hits) that mildly elevated p53 without significant mitotic defects, indicating that the stopwatch synergizes with mild p53 activation to halt proliferation. Mild p53 elevation lowered the stopwatch complex threshold for daughter cell arrest and slightly prolonged mitosis. Integrated over successive divisions, the cumulative effect of multiple short mitotic extensions triggered stopwatch-dependent arrest. Thus, the mitotic stopwatch endows the p53 network with a durable lineage memory of modest stress, explaining its tumor-suppressive role.

cell biology↗

bioRxiv2025

Longitudinal Changes in Cortical Response Dynamics with Deep Brain Stimulation to the Subcallosal Cingulate for Treatment-Resistant Depression

Deep brain stimulation (DBS) of the subcallosal cingulate cortex (SCC) is a promising intervention for treatment-resistant depression, yet objective biomarkers that track recovery remain limited. This study examined longitudinal changes in stimulation-evoked potentials (SEPs) to characterize how SCC-driven cortical communication evolves during treatment. Ten patients across three SCC-DBS trials underwent high-density EEG recordings at 4 and 24 weeks. SEP features were extracted from source-localized SCC signals and related to clinical outcomes and fractional anisotropy (FA) of midcingulate cingulum. Cortical responses showed a consistent reduction in latency and an increase in magnitude over time, indicating faster and stronger electrocortical signaling with chronic stimulation. Higher baseline midcingulate FA predicted greater latency acceleration, linking SEP timing to white-matter integrity. These findings identify temporal SEP dynamics as candidate mechanistic biomarkers that reflect circuit engagement during SCC-DBS and offer a pathway toward physiology-guided optimization of neuromodulation for depression. HighlightsO_LIReduced latency of stimulation evoked potential after 6 months of DBS C_LIO_LILarger cortical evoked response to single pulse perturbation after 6 months of DBS C_LIO_LIBaseline myelin integrity in MCC predicts magnitude of change in EP latency C_LI

neuroscience↗

bioRxiv2025

Phosphorylation remodels the mitotic centrosome matrix to generate bipartite gamma-tubulin complex docking sites

Mitotic centrosomes consist of centrioles surrounded by a proteinaceous matrix that docks and activates {gamma}-tubulin complexes ({gamma}TuCs) to nucleate microtubules for spindle assembly. During mitotic entry, phosphorylation at centrosomes remodels CDK5RAP2 family matrix proteins to generate {gamma}TuC docking sites. We address the mechanism of this conversion using C. elegans SPD-5 as a model. We show that SPD-5 contains two regions, PRGB1 and PRGB2, that are each sufficient for Polo-Like Kinase 1 (PLK1) phosphorylation-regulated {gamma}TuC binding. We define key phosphosites in each region and uncover autoinhibition mediated by interactions within and between them. PRGB2 is dimeric and requires {gamma}TuCs containing the Mozart family microprotein MZT-1 for binding, whereas PRGB1 is monomeric and binds independently of MZT-1. Our results support PLK1 phosphorylation inducing a conformational change that enables MZT-1-dependent PRGB2 binding, which in turn relieves PRGB1 inhibition. Such a multi-step mechanism would ensure robust spindle assembly by restricting microtubule nucleation in space and time.

cell biology↗

bioRxiv2025

Seipin mediates Perilipin-1 recruitment to lipid droplets to preserve human adipocyte identity

Congenital generalized lipodystrophy type 2 (CGL2) is caused by mutations in the BSCL2 gene, which encodes the protein seipin. However, how seipin loss causes adipose tissue failure remains unclear. Using human adipocyte progenitor cells capable of robust differentiation in vitro and in vivo, we reveal two unexpected findings that redefine CGL2 pathogenesis. First, seipin is dispensable for lipid droplet biogenesis but essential for recruiting the major adipocyte scaffold protein Perilipin 1 (PLIN1) to the lipid droplet surface. Second, we discover that the integrity of the lipid droplet serves as an organelle to nucleus quality control checkpoint enforcing adipocyte identity. Without seipin-dependent PLIN1 recruitment, adipocytes exhibit enhanced lipolysis and ceramide accumulation, triggering an unexpected cellular response of de-differentiation into a progenitor-like state. From this de-differentiated state, cells can undergo additional cycles of differentiation and de-differentiation upon repeated adipogenic stimuli. However, some cells escape de-differentiation, instead forming a single large droplet and displaying severe cellular structural abnormalities. Consistent with this model, we find functional adipose tissue can form in vivo from seipin-deficient cells, yet ultimately fails. These findings resolve conflicting models of CGL2 pathogenesis by reframing seipin as a regulator of PLIN1 recruitment, rather than droplet formation per se, and reveal the fundamental role of lipid droplet integrity in the development of functional human adipocytes.

cell biology↗

bioRxiv2025

A chromatin-associated pool of Aurora A controls kinetochore-microtubule attachments to ensure chromosome biorientation

Accurate chromosome segregation requires dynamic kinetochore-microtubule attachments that, under the regulation of Aurora family kinases, biorient and align replicated chromosomes. In C. elegans, Aurora A acts with the TPX2-related activator TPXL-1 to regulate these attachments and control spindle length. We show that, in addition to prominent spindle pole localization, TPXL-1-AurA has a chromatin-associated pool positioned between the sister kinetochores. Structural modeling and biochemical analysis support TPXL-1 directly recognizing the nucleosome acidic patch via an arginine anchor. Disrupting this interaction selectively removed chromatin-bound TPXL-1-AurA and caused chromosome missegregation, whereas elevation of the chromatin pool disrupted chromosome alignment. These opposing perturbations inversely affected kinetochore recruitment of the microtubule-binding Ska complex. These results support spatially distinct TPXL-1-AurA populations acting sequentially, with the spindle pole pool controlling spindle length by switching kinetochores out of a depolymerization-coupled state, and the chromatin pool controlling attachment stabilization to ensure biorientation prior to anaphase.

cell biology↗

bioRxiv2025

Community Challenge towards Consensus on Characterization of Biological Tissue: C4Bios First Findings

This study investigates methodological variability across various expert laboratories worldwide, with regards to characterizing the mechanical properties of biological tissues. Two testing rounds were conducted on the specific use case of uniaxial tensile testing of porcine aorta. In the first round, 24 labs were invited to apply their established methods to assess inter-laboratory variability. This revealed significant methodological diversity and associated variability in the stress-stretch results, underscoring the necessity for a standardized approach. In the second round, a consensus protocol was collaboratively developed and adopted by 19 labs in an attempt to minimize variability. This involved standardized sample preparation and uniformity in testing protocol, including the use of a common cutting and thickness measurement tool. Despite protocol harmonization, significant variability persisted across labs, which could not be solely attributed to inherent biological differences in tissue samples. These results illustrate the challenges in unifying testing methods across different research settings, underlining the necessity for further refinement of testing practices. Enhancing consistency in biomechanical experiments is pivotal when comparing results across studies, as well as when using the resulting material properties for in silico simulations in medical research.

bioengineering↗

bioRxiv2025

Patient-Derived Three-Dimensional Lung Tumor Models to Evaluate Response to Immunotherapy

Novel preclinical models that better mimic the in vivo tumor microenvironment are needed for advanced understanding of tumor biology and resistance/response to therapy. Herein, we report development of a novel ex vivo patient-derived three-dimensional lung tumor model (3D-LTM), that maintains features of human extracellular matrix, cell-cell interactions, and tissue architecture to evaluate a rapid response to immune checkpoint inhibitors (ICI). Within this model system, we recapitulated the heterogeneity of response to immunotherapy observed in non-small cell lung cancer (NSCLC) patients and defined signatures associated with response for predicting early response of ICI in patients. Spatial transcriptomics of the 3D-LTMs identified positive correlation of CD8+ T cell populations, CD4+ memory T cells, mast cells, NK cells, endothelial cells and non-classical monocytes with response status, whereas macrophages negatively correlated with response status. Pathway analysis of gene expression showed that chemokine signaling related pathways were activated in responder 3D-LTM tissues, whereas suppression of antigen presentation-related pathways and activation of Treg differentiation-related pathways was associated with 3D-LTMs that were not considered responders. This model system has utility for rapid testing of novel immune directed therapy outcomes and for developing biomarkers of ICI response in NSCLC.

cancer biology↗

bioRxiv2025

Pre-Clinical Evaluation of a Novel Immunomodulator: a potential immunotherapy for coronaviral disease

At present, no unifying animal study to quantify the effect of PI on innate immunity in animals has been reported. This study quantifies the effect of PI in mice and THP-1 cells using a multi-disciplinary approach employing flow cytometry to measure immune function and mass spectroscopy to identify metabolomic profiles of treated cells. For proof of principle, a case study was conducted to examine the potential benefit of administering PI to improve outcomes of a feline leukemia-positive cat that also contracted the effusive form of FIP. Our results indicate that quantifiable cellular and metabolic markers result from PI treatment and can be used to establish a platform measuring the efficacy of PI in modulating the innate immune system.

cell biology↗

bioRxiv2024

The CYK-4 GAP domain regulates cortical targeting of centralspindlin to promote contractile ring assembly and facilitate ring dissolution

During cytokinesis, an equatorial contractile ring partitions the cell contents. Contractile ring assembly requires an equatorial zone of active GTP-bound RhoA generated by the guanine nucleotide exchange factor ECT21,2. ECT2 is activated by centralspindlin, a complex composed of two molecules each of kinesin-6 and CYK4. During anaphase, Centralspindlin is activated at the central spindle between the separating chromosomes and diffuses to the plasma membrane, where it engages with ECT2 via its N-terminal half3,4. The C-terminal half of CYK4 contains a lipid-binding C1 domain that contributes to plasma membrane targeting5 and a GTPase-activating protein (GAP) domain that has an interaction surface for a Rho family GTPase, whose functions have remained unclear 1,3,4,6,7. Here, using the one-cell stage C. elegans embryo as a model, we show that RhoA and the Rho-binding interface of the CYK4 GAP domain drive the recruitment of centralspindlin to the equatorial cortex. By contrast, a point mutant that selectively disrupts GAP activity does not prevent cortical centralspindlin recruitment but instead substantially delays dissipation of centralspindlin from the cortex. These findings suggest that positive feedback, in which centralspindlin recruitment promotes the generation of active RhoA and active RhoA drives centralspindlin recruitment, is central to the rapid assembly of the contractile ring within a narrow time window. They also indicate that the CYK4 GAP catalytic activity contributes to release of centralspindlin from the cortex, potentially to ensure timely dissolution of the contractile ring.

cell biology↗

bioRxiv2024

Hybrid incompatibility emerges at the one-cell stage in interspecies Caenorhabditis embryos

Intrinsic reproductive isolation occurs when genetic divergence between populations disrupts hybrid development, preventing gene flow and enforcing speciation.1-4 Over the past two decades, researchers have identified molecular mechanisms underlying a few dozen cases of hybrid incompatibility in animals.5 Much of this work has focused on mismatches in zygotic gene regulation,6-11 but other mechanisms have also emerged, including symbiont-driven incompatibilities,12 nucleoporin mismatches affecting nuclear-cytoplasmic transport,13 and divergence in centromeric or heterochromatic regions and their regulatory proteins which can lead to the inability of the oocyte cytoplasm to segregate sperm-derived chromosomes.14-19 Since studies to date have focused on a limited number of species, uncovering mechanisms across diverse taxa will be important to understanding broader patterns of hybrid incompatibility. Here, we investigate the mechanistic basis of hybrid incompatibility in Caenorhabditis nematodes by leveraging the ability of C. brenneri females to produce embryos after mating with males from several other species. We find that incompatibilities emerge between fertilization and the onset of zygotic transcription, which begins at the 4-cell stage.20-23 In Caenorhabditis embryos, as in many animals,24,25 sperm deliver chromatin and centrioles into the oocyte.26-29 After remaining quiescent during oocyte meiosis, the sperm chromatin acquires a nuclear envelope, and centrioles initiate centrosome formation.30-32 Centrosomes remain tethered to the sperm pronucleus, which positions them near the cortex to establish anterior-posterior polarity.33,34 We identify two key processes that are destabilized in hybrids: (1) the ability of oocytes to control sperm-derived pronuclear expansion, and (2) successful polar body formation. When sperm pronuclear expansion is delayed, centrosomes detach, which leads to defects in polarity establishment. Hybrid embryos typically experience one or more stochastic failures of early developmental events that accumulate and eventually kill them.

evolutionary biology↗

bioRxiv2024

TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37s TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation. TRIM proteins form anti-parallel coiled-coil dimers with RING-B-box domains on each end. Oligomerization due to RING-RING interactions and conformational regulation by B-box-2-B-box-2 interfaces are critical for TRIM37 to suppress centrobin condensate formation. These results indicate that, analogous to anti-viral TRIM ligases, TRIM37 activation is linked to the detection of oligomerized substrates. Thus, TRIM37 couples peptide motif recognition and substrate-dependent oligomerization to effect ubiquitination-mediated clearance of ectopic centrosomal protein assemblies.

cell biology↗

bioRxiv2024

Patient-derived glioblastoma organoids as real-time avatars for assessing responses to clinical CAR-T cell therapy

Patient-derived tumor organoids have been leveraged for disease modeling and preclinical studies, but rarely applied in real-time to aid with interpretation of patient treatment responses in clinics. We recently demonstrated early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor T cells (EGFR-IL13R2 CAR-T cells) in patients with recurrent glioblastoma. Here we analyzed six sets of patient-derived glioblastoma organoids (GBOs) treated concurrently with the same autologous CAR-T cell products as patients in our phase 1 study. We found that CAR-T cell treatment led to target antigen reduction and cytolysis of tumor cells in GBOs, the degree of which correlated with CAR-T cell engraftment detected in patients cerebrospinal fluid (CSF). Furthermore, cytokine release patterns in GBOs mirrored patient CSF samples over time. Our findings highlight a unique trial design and GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and insights into immunotherapy efficacy.

cancer biology↗

bioRxiv2024

Mutual dependence between membrane phase separation and bacterial division protein dynamics in synthetic cell models

Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid-cell or the cell poles. An interesting result of such membrane-lipid interplay is the reorganization of lipid domains together with membrane-bound proteins at the onset of cell division, suggesting a functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinCDE and FtsZ as an archetypal spatial patterning system, we present a simple vesicle-based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, MinDE exhibit preferential binding and macro-scale pattern formation at Ld domains, which leads to altered oscillation mode selection in phase-separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase-separated GUVs leads to blebbing-like membrane deformations followed by the reorganization of Lo domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.

synthetic biology↗

bioRxiv2024

PPARγ activation by lipolysis-generated ligands is required for cAMP dependent UCP1 induction in human thermogenic adipocytes

ObjectiveThe uncoupling protein 1 (UCP1) is induced in brown or "beige" adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPAR{gamma} agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously. MethodsAdipocytes generated from primary human progenitor cells were differentiated without exposure to PPAR{gamma} agonists, and treated for 3, 6 or 78 hours to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses. ResultsUCP1 mRNA expression was induced within 3 hours of exposure to either Rsg or Fsk, indicating that Rsgs effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2, the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPAR{gamma} agonists. ConclusionUCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPAR{gamma}-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPAR{gamma} in response to cAMP occurs as a result of the production of natural PPAR{gamma} activating ligands through ATGL-mediated lipolysis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/607465v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18675a5org.highwire.dtl.DTLVardef@95cb66org.highwire.dtl.DTLVardef@5cb189org.highwire.dtl.DTLVardef@18bac05_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1/BUB-3 complex to C. elegans kinetochores

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

cell biology↗

bioRxiv2023

A distinct class of hematopoietic stem cells develop from the human yellow bone marrow

During aging, adipose tissue within the bone marrow expands while the trabecular red marrow contracts. The impact of these changes on blood cell formation remains unclear. To address this question, we performed single-cell and single-nuclei transcriptomic analysis on adipose-rich yellow bone marrow (BMY) and adipose-poor trabecular red marrow (BMR) from human subjects undergoing lower limb amputations. Surprisingly, we discovered two distinct hematopoietic niches, in which BMY contains a higher number of monocytes and progenitor cells expressing genes associated with inflammation. To further investigate these niches, we developed an in-vitro organoid system that maintains features of the human bone marrow. We find cells from BMY are distinct in their expression of the leptin receptor, and respond to leptin stimulation with enhanced proliferation, leading to increased monocyte production. These findings suggest that the age-associated expansion of bone marrow adipose tissue drives a pro-inflammatory state by stimulating monocyte production from a spatially distinct, leptin-responsive hematopoietic stem/progenitor cell population. SignificanceThis study reveals that adipose tissue within the human bone marrow is a niche for hematopoietic stem and progenitor cells that can give rise to pro-inflammatory monocytes through leptin signaling. Expansion of bone marrow adipose tissue with age and stress may thus underlie inflammageing.

developmental biology↗