Biology subjects

Audhya, A.

Publications and source records attributed to Audhya, A..

10 recordsLinked to original sources

bioRxiv2025

Multi-scale Molecular Imaging of Human Cells reveals COPI and COPII Vesicles at ER Exit Sites

Trafficking of secretory proteins from the endoplasmic reticulum (ER) to the Golgi apparatus comprises the first, essential steps toward the appropriate localisation of 30% of eukaryotic proteins. Coat protein complexes COPII and COPI are involved in forward and retrograde transport of cargo and cargo receptors between the ER and the Golgi, respectively. Although COPII forms coated vesicles in vitro, the biogenesis, morphology and organisation of transport carriers in mammalian cells is debated. Here we use in situ cryo-electron tomography and super-resolution fluorescence microscopy to reveal the molecular architecture of ER exit sites in human cells that were not perturbed with drugs, temperature blocks, or overexpression systems. We visualise ribosome-exclusion zones enriched with COPII and COPI-coated vesicles and thus resolve the debate regarding the existence of COPII coated vesicles. COPII vesicles derive from ER membranes, whereas COPI vesicles originate from vesicular-tubular clusters that constitute the ER-Golgi intermediate compartment (ERGIC). We quantify coated vesicle morphology and positioning with respect to other ER exit site components, providing a molecular description of the organisation of the mammalian early secretory pathway.

cell biology↗

bioRxiv2025

A privileged ER compartment for post-translational heteromeric assembly of an ion channel

Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric complexes are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-a-go-go related gene (hERG) is essential for the normal function of cardiac IKr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome (LQTS). While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, which depend on the presence of an N-terminal "RXR" ER retention signal, represent a privileged ER sub-compartment distinct from that containing ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurred by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly post-translationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly. Significance StatementhERG potassium channels are essential for repolarizing the ventricular action potential. Heteromeric hERG channels, which conduct cardiac IKr, are formed by association of hERG1a and 1b subunits. However, mechanisms governing the assembly of hERG and other heteromeric protein complexes are poorly understood. We identify a noncanonical ER compartment that sequesters hERG1b, distinct from previously described ER quality control pathways that degrade misfolded proteins, including LQTS-associated hERG variants. Instead, this compartment preserves hERG1b and facilitates its post-translational assembly with hERG1a, challenging the current view of simultaneous, cotranslational heteromeric assembly. Our findings redefine ER retention as an active regulatory step in protein complex formation that may broadly govern hetero-oligomeric assembly in many systems and facilitate therapeutic approaches to arrhythmia and other diseases.

cell biology↗

bioRxiv2024

Multiple roles for TFG ring complexes in neuronal cargo trafficking

Pathological variants in Trk-fused gene (TFG) have been implicated in a variety of neurodegenerative conditions. In particular, mutations within its amino-terminal PB1 domain have been suggested to cause hereditary spastic paraplegia (HSP), resulting in progressive lower limb spasticity and weakness. The structural basis for this effect is unknown. Here, we combine X-ray crystallography and cryo-electron microscopy to determine a structural model of TFG, demonstrating the mechanism by which it forms octameric ring complexes. A network of electrostatic and hydrophobic interactions defines the interface between protomers. Moreover, we show that mutations identified previously in HSP patients disrupt this interface, destabilizing octamers, which ultimately leads to axonopathy. Surprisingly, the impacts of these variants are not equivalent in vivo, highlighting the existence of multiple, distinct mechanisms by which TFG mutations contribute to neurodegenerative disease.

biochemistry↗

bioRxiv2024

Long term rescue of Alzheimer deficits in vivo by one-time gene-editing of App C-terminus.

Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can manipulate upstream pathophysiologic events and alter disease-course in wider patient populations. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein (App), relevant for Alzheimers disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus in mouse and human neurons, while preserving N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway; inhibiting toxic APP-beta-cleavage fragments (including Abeta) and upregulating neuroprotective APP-alpha-cleavage. AAV-editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months with no detectable abnormalities in germline-edited WT mice, and pathologic alterations in glial-transcriptomes are also normalized. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and this upstream manipulation is expected to be effective for all forms of AD.

neuroscience↗

bioRxiv2024

Toxoplasma gondii infection accelerates the progression of hereditary spastic paraplegia

The parasitic protozoa Toxoplasma gondii chronically infects the central nervous system of an estimated one-third of the human population. Infection is generally subclinical, but immunocompromised individuals can experience a variety of neurological symptoms. Meta-analyses of T. gondii seropositivity have suggested a correlation between T. gondii infection and neurologic disease. While mechanistic studies on the relationship between T. gondii infection and neurologic disease have been attempted in mice, mice are particularly susceptible to T. gondii, making them an effective model for investigating mechanisms of infection, but not ideal for examining the relationship between long-term chronic T. gondii infection and neurologic disease. Rats more closely mimic human clearance of T. gondii after acute infection, but a lack of rat models of neurologic disease has limited studies on the interplay between T. gondii infection and neurologic disease progression. We have employed a previously characterized rat model of a complex form of hereditary spastic paraplegia (HSP), a class of neurodegenerative disorders which cause axonal degeneration and lower limb spasticity, in order to assess the effect of chronic T. gondii infection on neurodegenerative disease. We find that infected rats with hereditary spastic paraplegia exhibit significantly exacerbated behavioral and neuromorphological HSP symptoms compared to uninfected HSP mutant rats, with little correlative effect in infected versus uninfected control animals. We further find that all infected rats regardless of genotype exhibit a robust immune response to T. gondii infection, effectively clearing the parasite below the limit of detection of multiple assays of parasitemia and exhibiting no detectable increase in neuroinflammation seven weeks post-infection. These results suggest that chronic undetected T. gondii infection may exacerbate neurodegenerative disease even in immunocompetent individuals and may contribute to neurodegenerative disease heterogeneity. Author SummaryThe long-term consequences of previous acute infections are poorly understood, but are becoming increasingly appreciated, particularly in the era of long Covid. Altered progression of other diseases later in life may be among the long-term consequences of previous infections. Here we investigate the relationship between previous infection with the parasite Toxoplasma gondii, which infects [~]30% of the global population, and neurodegenerative disease using a rat model of hereditary spastic paraplegia (HSP). We find that previous infection with T. gondii accelerates motor dysfunction in HSP rats, despite robust clearance of the parasite by infected rats. Our results suggest that previously cleared infections may alter the progression of other diseases later in life and contribute to neurodegenerative disease heterogeneity.

microbiology↗

bioRxiv2024

Delineating the shape of COPII coated membrane bud

Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. Our recent study on the COPII protein Sar1 showed that the inserted volume of the protein into the membrane determines the degree of membrane bending. However, it is unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological dissimilarities, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first demonstrate the membrane bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of inter-peptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG (MARTINI) simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using Dynamical Triangulated Surface (DTS) simulations based on the Helfrich Hamiltonian we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, we show that Sec23 and Sec24 act as a spacer to preserve a dispersed arrangement of Sar1 protomers and to help determine the overall shape of the membrane bud.

biophysics↗

bioRxiv2023

Defining Cardiac Nerve Architecture During Development, Disease, and Regeneration

Cardiac nerves regulate neonatal mouse heart regeneration and are susceptible to pathological remodeling following adult injury. Understanding cardiac nerve remodeling can lead to new strategies to promote cardiac repair. Our current understanding of cardiac nerve architecture has been limited to two-dimensional analysis. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling tools to define cardiac nerve architecture and neurovascular association during development, disease, and regeneration. Our results demonstrate that cardiac nerves sequentially associate with coronary veins and arteries during development. Remarkably, our results reveal that parasympathetic nerves densely innervate the ventricles. Furthermore, parasympathetic and sympathetic nerves develop synchronously and are intertwined throughout the ventricles. Importantly, the regenerating myocardium reestablishes physiological innervation, in stark contrast to the non-regenerating heart. Mechanistically, reinnervation during regeneration is dependent on collateral artery formation. Our results reveal how defining cardiac nerve remodeling during homeostasis, disease, and regeneration can identify new therapies for cardiac disease.

developmental biology↗

bioRxiv2022

Molecular mechanism of GTP binding- anddimerization-induced enhancement ofSar1-mediated membrane remodeling

The Sar1 GTPase initiates Coat Protein II (COPII)-mediated protein transport by generating membrane curvature at subdomains on the Endoplasmic Reticulum, where it is activated by the guanine nucleotide exchange factor (GEF) Sec12. Crystal structures of GDP- and GTP-bound forms of Sar1 suggest that it undergoes a conformational switch in which GTP binding enhances the exposure of an amino-terminal amphipathic helix necessary for efficient membrane penetration. However, key residues in the amino-terminus were not resolved in crystal structures, and experimental studies have suggested that the amino-terminus of Sar1 is solvent-exposed in the absence of membrane, even in the GDP-bound state. Therefore, the molecular mechanism by which GTP binding activates the membrane remodeling activity of Sar1 remains unclear. Using atomistic molecular dynamics simulations, we compare the membrane binding and curvature generation activities of Sar1 in its GDP- and GTP-bound states. We show that in the GTP-bound state, Sar1 inserts into the membrane with its complete (residues 1-23) amphipathic amino-terminal helix, while Sar1-GDP binds to the membrane only through its first 12 residues. Such differential membrane binding modes translate into significant differences in the protein volume inserted into the membrane. As a result, Sar1-GTP generates positive membrane curvature 10-20 times higher than Sar1-GDP. Dimerization of the GTP-bound form of Sar1 further amplifies curvature generation. Taken together, our results present a detailed molecular mechanism for how the nucleotide-bound state of Sar1 regulates its membrane binding and remodeling activities in a concentration dependent manner, paving the way toward a better understanding COPII-mediated membrane transport. Significance StatementAmphipathic helices play established roles in penetrating lipid bilayers to produce local membrane curvature. The degree of curvature generated has been suggested to depend on the penetration depth of the amphipathic helix, its amino acid sequence, and the protein volume inserted into the membrane. However, the relative contributions of each of these factors in membrane bending remain unclear. Using the Sar1 protein monomer bound to different nucleotides (GDP vs. GTP) and its GTP-bound dimeric form as examples, we explicitly show that while the precise amino acid sequence and insertion depth of the amphipathic helix are relevant, the generated membrane curvature is most correlated with the volume of protein insertion into the membrane.

biophysics↗

bioRxiv2021

TES-1/Tes protects junctional actin networks under tension from self-injury during epidermal morphogenesis in the C. elegans embryo

During embryonic morphogenesis, the integrity of epithelial tissues depends on the ability of cells in tissue sheets to undergo rapid changes in cell shape while preventing self-injury to junctional actin networks. LIM domain-containing repeat (LCR) proteins are recruited to sites of strained actin filaments in cultured cells [1-3], and are therefore promising candidates for mediating self-healing of actin networks, but whether they play similar roles in living organisms has not been determined. Here, we establish roles for Caenorhabditis elegans TES-1/Tes, an actin-binding LCR protein present at apical junctions, during epithelial morphogenesis. TES-1::GFP is recruited to apical junctions during embryonic elongation, when junctions are under tension; in embryos in which stochastic failure of cell elongation occurs, TES-1 is only strongly recruited to junctions in cells that successfully elongate, and recruitment is severely compromised in genetic backgrounds in which cell shape changes do not successfully occur. tes-1 mutant embryos display junctional F-actin defects, and loss of TES-1 strongly enhances tension-dependent injury of junctional actin networks in hypomorphic mutant backgrounds for CCC components, suggesting that TES-1 helps to prevent self-injury of junctional actin networks during rapid cell shape change. Consistent with such role, a fragment of TES-1 containing its LIM domains localizes to stress fiber strain sites (SFSS) in cultured vertebrate cells. Together, these data establish TES-1 as a tension-sensitive stabilizer of the junctional actin cytoskeleton during embryonic morphogenesis.

developmental biology↗

bioRxiv2019

Local regulation of lipid synthesis controls ER sheet insertion into nuclear envelope holes to complete nuclear closure

The nuclear permeability barrier depends on closure of holes in the nuclear envelope (NE). Here, we use meiotic C. elegans oocytes to demonstrate that local control of glycerophospholipid synthesis by CNEP-1/CTDNEP1 regulates the insertion of ER sheets into NE holes and functions independently of ESCRT-III to ensure NE closure. Deletion of CNEP-1 causes excess incorporation of ER membranes into NE holes and a defective NE permeability barrier. ESCRT-III components accumulate at the NE opening surrounding the meiotic spindle, and loss of NE adaptors for ESCRT-III exacerbates NE sealing defects in cnep-1 mutants. Limiting ER sheet production by restoring glycerophospholipid synthesis in cnep-1 mutants rescued NE permeability defects. 3D analysis showed that membrane sheets feed into and narrow NE holes occluded by meiotic spindle microtubules supporting a role for ER sheet insertion in NE closure. Thus, feeding of ER sheets into NE holes must be coordinated with production of ER sheets near the NE to promote NE closure.

cell biology↗