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Biology subjects

Nanda, S.

Publications and source records attributed to Nanda, S..

At least 19 recordsLinked to original sources

Neural subtypes in developmental stuttering

Developmental stuttering is a complex neurodevelopmental disorder characterized by disfluent speech. At the individual level, the behavioral manifestations of stuttering vary considerably, likely reflecting heterogeneity in underlying neural mechanisms. In this study, we examined individual-specific differences in the brains of children who stutter (CWS), by implementing normative modeling, a framework that quantifies how an individual deviates from an age- and sex-matched reference population. We applied this approach to identify individual-specific structural brain atypicalities using gray and white matter volumes. These volumes were derived from MRI scans from a large mixed-longitudinal dataset of 235 and 240 scans from CWS and fluent controls respectively, aged between 3 and 12 years. Individual deviation maps capturing these atypicalities were then used to cluster CWS into subtypes based on similarities in their neuroanatomical profiles. This analysis identified four neural subtypes with distinct neuroanatomical atypicalities relative to fluent controls. The key findings were a basal ganglia-thalamo-cerebellar subtype associated with higher stuttering severity and lower rates of recovery, and a white matter subtype characterized by mild severity and a higher likelihood of recovery. The remaining two subtypes showed cerebellar differences alongside alterations in brain regions involved in sensorimotor integration. Moreover, cerebellar volume atypicalities were present in all four subtypes, indicating that cerebellar alterations were present across otherwise distinct neural profiles and may represent a shared neuroanatomical feature of stuttering. These findings indicate that examining individual-specific neural differences and subtyping based on patterns of neural atypicalities provides valuable insight into the heterogeneity of developmental stuttering and represents a promising direction for improving our understanding of the disorder.

neuroscience↗

Unique pore architecture underlies constitutive gating of human retinalTRPM1

Transient receptor potential melastatin 1 (TRPM1), a Ca{superscript 2}-permeable nonselective cation channel essential for retinal ON bipolar cell signaling and night vision, and implicated in congenital night blindness, has remained structurally and functionally poorly characterized. Here we report the first cryo-electron microscopy structure of human TRPM1 in conducting state. Although the channel assembles as a tetramer, it adopts an unexpected clockwise domain-swapped pore module with rotational geometry inverse to that observed in previously characterized 6-TM tetrameric channels. This inverted topology is accompanied by extensive remodeling of the S5-P-S6 module, dilation of the selectivity filter, expansion of the central cavity, and splaying of S6 to form a wide intracellular gate. Single-channel recordings reveal constitutive activity consistent with the conductive state captured. Together, these findings uncover a new 6-TM fold in tetrameric channel and provide a framework for understanding TRPM1 gating and disease-associated dysfunction.

biochemistry↗

Rnd3 regulates cell morphodynamics by spatial restriction of cell contraction signaling

Cell migration is enabled by dynamic changes in cell shape, which are controlled by spatio-temporal activity patterns of the Rho GTPases Rac1 and RhoA. Classical models proposed that these activity patterns are generated by mutual inhibition between the front signal Rac1 and the back signal RhoA, leading to opposing gradients that define the direction of cell migration. However, direct measurements of signal crosstalk showed that Rac1 can activate RhoA, which is incompatible with mutual inhibition. Furthermore, opposing Rac1 and RhoA gradients generated by mutual inhibition would need to overlap at least partially in the cell center. In contrast, both Rac and Rho activities were largely absent in the cell center, and both found to be highly localized near the cell edge in the cell periphery. Here, we hypothesized that these spatio-temporal Rho GTPase activity patterns are generated by the mutual inhibition between RhoA and the unconventional Rho family member Rnd3. Using rapid, optogenetic and chemical perturbations, we confirmed this mutual inhibitory crosstalk. However, we found that this crosstalk does not lead to the expected mutually exclusive spatio-temporal patterns of Rnd3 activity and cell retraction in spontaneously migrating, unperturbed cells. Instead, Rnd3 activity was even slightly elevated during cell retraction and was surprisingly depleted during cell protrusion. We discovered that this depletion is caused by the inhibition of Rnd3 by Rac1 activity, and that this newly identified inhibition is much more pronounced compared to the inhibition of Rnd3 by Rho. By combining rapid optogenetic perturbations with pharmacological manipulations, we found that Rac1 inhibition by Rnd3 is mediated by p21-activated kinases (PAKs). Investigations into the function of Rnd3 showed that it is required for the tight spatio-temporal regulation of the cell contraction/retraction signal Rho, and that it prevents ectopic, highly dynamic, spontaneous Rho activity pulses within the whole cell attachment area. Interestingly, protrusion-retraction dynamics were also severely inhibited in the absence of Rnd3, and its overexpression stimulated this process. Taken together, we show that Rac and not Rho acts as the major Rnd3 inhibitor in cells. Furthermore, our findings support a mechanism, in which Rnd3 acts as a global inhibitor of Rho that spatially restricts Rho activity to regions near the edge of migrating cells.

cell biology↗

Sustained quenching not always means photoinhibition

Photosynthetic light harvesting complexes (LHC) are involved in light absorption and energy dissipation. By modulating the photosystems absorption cross section, they affect their photosynthetic activity and non-photochemical quenching (NPQ) capacity. These processes have been widely studied by spectrally integrated chlorophyll fluorescence methods, which mask their associated spectral information. We explored in aspen and Arabidopsis npq mutants how the absence of these components affects the development of NPQ spectra under two contrasting conditions: in the absence and presence of photoinhibition. We proposed a new parameter to estimate the development of new emitting species (NESD) during time-spectrally resolved NPQ inductions and a pipeline to disentangle PSII energy partitioning heterogeneity. We demonstrate that LHCB, PsbS and zeaxanthin is required for NESD. By combining gas exchange with spectrally resolved kinetics, we show that under photoinhibitory conditions, however, NES develops in the absence of PsbS and zeaxanthin, and the resulting sustained quenching occurring independently of photoinhibition. Furthermore, we found that in the absence of LHCB and Curvature Thylakoid 1 a significant increase in photoinhibition was observed. This suggest that in the long term effective photoprotection requires the presence of LHCB and thylakoid plasticity, while PsbS and zeaxanthin play a major role in catalyzing LHCII-dependent quenching.

plant biology↗

Longitudinal multi-omic profiling uncovers immune escape and predictors of response in multiple myeloma

Multiple myeloma (MM) is an incurable malignancy of clonally expanded plasma cells shaped by complex interactions with the immune microenvironment. To investigate immune factors driving treatment response and resistance, we conducted multi-omics profiling including CD138neg single-cell RNA sequencing of 243 bone marrow samples from 102 patients (631,226 cells) and CD138pos bulk RNA and whole-genome sequencing from 209 samples. Longitudinal analyses revealed that interferon gamma signaling impairs T cell memory after autologous stem cell transplant, while naive B cell abundance and immunoglobulin diversity correlated with improved progression-free survival (HR = 0.48, p = 2.3e-4). At disease progression, MM cells upregulated cancer-testis antigens and immune effector genes, with concurrent B cell depletion, enrichment of myeloid-derived suppressor cell genes in monocytes, and T cell exhaustion. These findings highlight dynamic immune-tumor interactions, identifying naive B cell reconstitution as a biomarker of durable response, and cancer-testis antigens as potential targets for high-risk disease at progression. Statement of SignificanceLongitudinal profiling of multiple myeloma and the immune microenvironment revealed dynamic immune-tumor interactions across the disease course. Dysfunctional CD8 T cells limited memory formation post-transplant, while naive B recovery associated with sustained treatment response. At progression, cancer-testis antigen expression associated with immunosuppression, revealing novel mechanisms of immune escape.

cancer biology↗

Crosstalk between Rac and Rap GTPases in migrating cells

To enable effective cell migration, local cell protrusion has to be coordinated with local cell attachment. Here, we investigate spatio-temporal activity patterns of key regulators of cell protrusion and adhesion, the small GTPases Rac and Rap, in migrating cells. These analyses show that Rac activity correlates very tightly with instantaneous cell protrusion events, while the Rap activity stays elevated for prolonged time periods after protrusion and is also detectable before cell protrusion. Direct analysis of activity crosstalk in living cells via light-based perturbation methods revealed that Rap can efficiently activate Rac, however, reciprocal crosstalk from Rac to Rap was not detectable. These findings suggest that Rap plays an instructive role in the generation of cell protrusions by its ability to activate Rac. Furthermore, prolonged Rap activity suggests that this molecule also plays a role in maintenance or stabilization of cell protrusions. Indeed, morphological analysis of Rap1-depleted A431 cells revealed a significant reduction of the cell attachment area, suggesting that Rap stimulated cell adhesion might indeed stabilize newly formed protrusions. Taken together, our study suggests a mechanism, by which cell protrusion is coupled to cell adhesion via unidirectional crosstalk that connects the activity of the small GTPases Rap and Rac.

cell biology↗

Worm Perturb-Seq: massively parallel whole-animal RNAi and RNA-seq

The transcriptome provides a highly informative molecular phenotype to connect genotype to phenotype and is most frequently measured by RNA-sequencing (RNA-seq). Therefore, an ultimate goal is to perturb every gene and measure changes in the transcriptome. However, this remains challenging, especially in intact organisms due to different experimental and computational challenges. Here, we present Worm Perturb-Seq (WPS), which provides high-resolution RNA-seq profiles for hundreds of replicate perturbations at a time in a living animal. WPS introduces multiple experimental advances that combine strengths of bulk and single cell RNA-seq, and that further provides an analytical framework, EmpirDE, that leverages the unique power of the large WPS datasets. EmpirDE identifies differentially expressed genes (DEGs) by using gene-specific empirical null distributions, rather than control conditions alone, thereby systematically removing technical biases and improving statistical rigor. We applied WPS to 103 Caenhorhabditis elegans nuclear hormone receptors (NHRs) to delineate a Gene Regulatory Network (GRN) and found that this GRN presents a striking pairwise modularity where pairs of NHRs regulate shared target genes. We envision that the experimental and analytical advances of WPS should be useful not only for C. elegans, but will be broadly applicable to other models, including human cells.

genomics↗

Spillover is the dominant non-photochemical quenching mechanism in angiosperms

Non-photochemical quenching (NPQ) is an important photoprotective process in plants, but all molecular details of the process(es) involved are yet not understood. We have used advanced spectroscopic techniques (including simultaneous time- and spectrally-resolved room-temperature chlorophyll fluorescence analysis and spectro-kinetic deconvolution) to analyse the processes in Arabidopsis, hybrid aspen and Scots pine plants. We used four well-characterized Arabidopsis lines (npq1, npq2, npq4 and L17) affected in NPQ, together with hybrid aspen lines with corresponding modifications that we generated. The data are described best by a model for NPQ induction with up to five fluorescence components representing distinct biochemical entities. A dominant fluorescing species at the end of NPQ induction was identified as functionally detached and quenched LHCII but most importantly we believe that one represents a "PSII-PSI (Photosystem II-Photosystem I) complex" where direct energy transfer between PSII and PSI (spillover) take place. This provides strong quenching in all three plant species. We suggest a new integrated model for NPQ in higher plants where spillover is a major element and suggest roles for PsbS and zeaxanthin. Moreover, we discuss the link between NPQ and thylakoid rearrangements as thylakoid destacking facilitates direct contact between PSII and PSI; a prerequisite for spillover.

plant biology↗

Dendritome Mapping Unveils Spatial Organization of Striatal D1/D2-Neuron Morphology

Morphology is a cardinal feature of a neuron and mediates its functions, but profiling neuronal morphologies at scale remains a formidable challenge. Here we describe a generalizable pipeline for large-scale brainwide profiling of dendritic morphology of genetically-defined single neurons in the mouse brain. We generated a dataset of 3,762 3D-reconstructed and reference-atlas mapped striatal D1- and D2-medium spiny neurons (MSNs). Integrative morphometric analyses reveal distinct impacts of D1/D2 and anatomical locations on MSN morphology. To analyze striatal regional features of MSN dendrites without prior anatomical constraints, we assigned MSNs to a lattice of cubic boxes in the reference brain atlas, and summarized morphometric representation ("eigen-morph") for each box and clustered boxes with shared morphometry. This analysis reveals 6 modules with characteristic dendritic features and spanning contiguous striatal territories, each receiving distinct corticostriatal inputs. Finally, we found aging confers robust and concordant dendritic length and branching defects in D1/D2-MSNs, while Huntingtons disease (HD) mice exhibit MSN-subtype and striatal regional specific pathology. Together, our study demonstrates a systems-biology approach to profile dendritic morphology of genetically-defined single-neurons; and defines novel striatal D1/D2-MSN morphological territories and aging- or HD-associated pathologies.

neuroscience↗

A single-cell atlas characterizes dysregulation of the bone marrow immune microenvironment associated with outcomes in multiple myeloma

Multiple Myeloma (MM) remains incurable despite advances in treatment options. Although tumor subtypes and specific DNA abnormalities are linked to worse prognosis, the impact of immune dysfunction on disease emergence and/or treatment sensitivity remains unclear. We established a harmonized consortium to generate an Immune Atlas of MM aimed at informing disease etiology, risk stratification, and potential therapeutic strategies. We generated a transcriptome profile of 1,149,344 single cells from the bone marrow of 263 newly diagnosed patients enrolled in the CoMMpass study and characterized immune and hematopoietic cell populations. Associating cell abundances and gene expression with disease progression revealed the presence of a proinflammatory immune senescence-associated secretory phenotype in rapidly progressing patients. Furthermore, signaling analyses suggested active intercellular communication involving APRIL-BCMA, potentially promoting tumor growth and survival. Finally, we demonstrate that integrating immune cell levels with genetic information can significantly improve patient stratification.

genomics↗

Gossamer: Scaling Image Processing and Reconstruction to Whole Brains

Neuronal reconstruction-a process that transforms image volumes into 3D geometries and skeletons of cells- bottlenecks the study of brain function, connectomics and pathology. Domain scientists need exact and complete segmentations to study subtle topological differences. Existing methods are diskbound, dense-access, coupled, single-threaded, algorithmically unscalable and require manual cropping of small windows and proofreading of skeletons due to low topological accuracy. Designing a data-intensive parallel solution suited to a neurons shape, topology and far-ranging connectivity is particularly challenging due to I/O and load-balance, yet by abstracting these vision tasks into strategically ordered specializations of search, we progressively lower memory by 4 orders of magnitude. This enables 1 mouse brain to be fully processed in-memory on a single server, at 67x the scale with 870x less memory while having 78% higher automated yield than APP2, the previous state of the art in performant reconstruction.

neuroscience↗

Local microtubule and F-actin distributions fully determine the spatial geometry of Drosophila sensory dendritic arbors

Dendritic morphology underlies the source and processing of neuronal signal inputs. Morphology can be broadly described by two types of geometric characteristics. The first is dendrogram topology, defined by the length and frequency of the arbor branches; the second is spatial embedding, mainly determined by branch angles and tortuosity. We have previously demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. Here we relate the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. We first reconstruct and analyze 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes. We observe that branches with higher microtubule concentration are overall straighter and tend to deviate less from the direction of their parent branch. F-actin displays a similar effect on the angular deviation from the parent branch direction, but its influence on branch tortuosity varies by class and genotype. We then create a computational model of dendritic morphology purely constrained by the cytoskeletal composition imaged from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors.

neuroscience↗

Crosstalk between Rac and Rho GTPase activity mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by a decrease in migration distance and an increase in migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.

cell biology↗

The Zinc-BED transcription factor Bedwarfed promotes proportional dendritic growth and branching through transcriptional and translational regulation in Drosophila

Dendrites are the primary points of sensory or synaptic inputs to a neuron and play an essential role in synaptic integration and neural function. Despite the functional importance of dendrites, relatively less is known about the underlying mechanisms regulating cell-type specific dendritic patterning. Herein, we have dissected functional roles of a previously uncharacterized gene, CG3995, in cell-type specific dendritic development in Drosophila melanogaster. CG3995, which we have named bedwarfed (bdwf), encodes a zinc-finger BED-type protein which is required for proportional growth and branching of dendritic arbors, exhibits nucleocytoplasmic expression, and functions in both transcriptional and translational cellular pathways. At the transcriptional level, we demonstrate a reciprocal regulatory relationship between Bdwf and the homeodomain transcription factor (TF) Cut. We show that Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins produced phenotypically similar dendritic hypotrophy defects as observed in bdwf mutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, our findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. Taken together, these results provide new insights into the complex, combinatorial and multi-functional roles of transcription factors (TFs) in directing diversification of cell-type specific dendritic development.

neuroscience↗

A Transcriptional Cofactor Regulatory Network for the C. elegans Intestine

Chromatin modifiers and transcriptional cofactors (collectively referred to as CFs) work with DNA-binding transcription factors (TFs) to regulate gene expression. In multicellular eukaryotes, distinct tissues each execute their own gene expression program for accurate differentiation and subsequent functionality. While the function of TFs in differential gene expression has been studied in detail in many systems, the contribution of CFs has remained less explored. Here we uncovered the contributions of CFs to gene regulation in the Caenorhabditis elegans intestine. We first annotated 366 CFs encoded by the C. elegans genome and assembled a library of 335 RNAi clones. Using this library, we analyzed the effects of individually depleting these CFs on the expression of 19 fluorescent transcriptional reporters in the intestine and identified 216 regulatory interactions. We found that different CFs interact specifically with different promoters, and that both essential and intestinally expressed CFs exhibit the highest proportion of interactions. We did not find all members of CF complexes acting on the same set of reporters but instead found diversity in the promoter targets of each complex component. Finally, we found that previously identified activation mechanisms for the acdh-1 promoter use different CFs and TFs. Overall, we demonstrate that CFs function specifically rather than ubiquitously at intestinal promoters and provide an RNAi resource for reverse genetic screens.

systems biology↗

Systems-level transcriptional regulation of Caenorhabditis elegans metabolism

Metabolism is precisely controlled to ensure organismal development and homeostasis. Several mechanisms regulate metabolism, including allosteric control and transcriptional regulation of metabolic enzymes and transporters. So far, metabolism regulation has mostly been described for individual genes and pathways, and the extent of transcriptional regulation of the entire metabolic network remains largely unknown. Here, we find that more than three-quarters of all metabolic genes are transcriptionally regulated in the nematode Caenorhabditis elegans. We find that many annotated metabolic pathways are coexpressed, and we use gene expression data and the iCEL1314 metabolic network model to define coregulated sub-pathways in an unbiased manner. Using a large gene expression compendium, we determine the conditions where sub-pathways exhibit strong coexpression. Finally, we develop WormClust, a web application that enables a gene-by-gene query of genes to view their association with metabolic (sub)-pathways. Overall, this study sheds light on the ubiquity of transcriptional regulation of metabolism and provides a blueprint for similar studies in other organisms, including humans.

systems biology↗

O2 photoreduction at acceptor side of Photosystem I provide photoprotection to conifer thylakoids in early spring

Green organisms evolve O2 via photosynthesis and consume by respiration. Net O2 consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles demonstrate strong O2 consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring. This phenomenon deviates from the general finding that photosynthetic organisms evolve O2 upon illumination. By using different electron transport chain inhibitors, we showed that O2 consumption occurred around photosystem (PS) I and correlated with higher abundance of flavodiiron (Flv) A protein in ES thylakoid membranes. Furthermore, by measuring P700 absorption changes, we separated different alternative electron flow pathways and demonstrated that electron scavenging from the acceptor-side of PSI via O2 photoreduction is a major alternative pathway in ES. This photoprotection mechanism in vascular plants indicates that conifers have developed an adaptative evolution trajectory for growing in harsh environments.

plant biology↗

D-2-Hydroxyglutarate dehydrogenase connects the propionate shunt to ketone body metabolism in Caenorhabditis elegans

In humans, mutations in D-2-hydroxyglutarate (D-2HG) dehydrogenase (D2HGDH) result in D-2HG accumulation, delayed development, seizures, and ataxia. While the mechanisms of 2HG-associated diseases have been studied extensively, the endogenous metabolism of D-2HG remains unclear in any organism. Here, we find that, in Caenorhabditis elegans, D-2HG is produced in the propionate shunt, which is transcriptionally activated when flux through the canonical, vitamin B12-dependent propionate breakdown pathway is perturbed. Deletion of the D2HGDH ortholog, dhgd-1, results in embryonic lethality, mitochondrial defects, and the upregulation of ketone body metabolism genes. Viability can be rescued by RNAi of hphd-1, which encodes the enzyme that produces D-2HG, or by supplementing either vitamin B12 or the ketone body 3-hydroxybutyrate (3HB). Altogether, our findings support a model in which C. elegans relies on ketone bodies for energy when vitamin B12 levels are low, and in which a loss of dhgd-1 causes lethality by limiting ketone body production. HIGHLIGHTS- D-2-hydroxyglutarate is produced by HPHD-1 in the propionate shunt pathway - DHGD-1 recycles 2-hydroxyglutarate to sustain flux through the propionate shunt - dhgd-1 loss perturbs ketone body metabolism and causes embryonic lethality - 3-Hydroxybutyrate, vitamin B12 or hphd-1 RNAi rescue dhgd-1 mutant lethality

biochemistry↗