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Postdoctoral Associate, Department of Cell Biology
Yale School of Medicine
Biophysics, Biochemistry, Neuroscience, Membrane Biology, Nuclear Biology, Mechanotransduction, Biomechanics
Sathish Ramakrishnan, Manindra Bera, Jeff Coleman, James E Rothman, and Shyam S Krishnakumar
eLife, eISSN: 2050084X, Pages: 1-18, Published: May 2020 eLife Sciences Publications, Ltd
Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free ‘peripheral’ SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.
Manindra Bera and Kaushik Sengupta
Nucleus, ISSN: 19491034, eISSN: 19491042, Pages: 99-110, Published: 1 January 2020 Informa UK Limited
ABSTRACT Nuclear lamins form an elastic meshwork underlying the inner nuclear membrane and provide mechanical rigidity to the nucleus and maintain shape. Lamins also maintain chromosome positioning and play important roles in several nuclear processes like replication, DNA damage repair, transcription, and epigenetic modifications. LMNA mutations affect cardiac tissue, muscle tissues, adipose tissues to precipitate several diseases collectively termed as laminopathies. However, the rationale behind LMNA mutations and laminopathies continues to elude scientists. During interphase, several chromosomes form inter/intrachromosomal contacts inside nucleoplasm and several chromosomal loops also stretch out to make a ‘loop-cluster’ which are key players to regulate gene expressions. In this perspective, we have proposed that the lamin network in tandem with nuclear actin and myosin provide mechanical rigidity to the chromosomal contacts and facilitate loop-clusters movements. LMNA mutations thus might perturb the landscape of chromosomal contacts or loop-clusters positioning which can impair gene expression profile.
Debdatto Mookherjee, Subhrangshu Das, Rukmini Mukherjee, Manindra Bera, Swadhin Chandra Jana, Saikat Chakrabarti, and Oishee Chakrabarti
Autophagy, ISSN: 15548627, eISSN: 15548635, Pages: 1-24, Published: 2020 Informa UK Limited
Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate "mitoplasts". This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The "mitoplasts" generated are degraded by reticulo-mito-phagy - simultaneously affecting dual organelle turnover.
Yale Journal of Biology and Medicine, ISSN: 00440086, Pages: 541-548, Published: 2019
Sathish Ramakrishnan, Manindra Bera, Jeff Coleman, Shyam S. Krishnakumar, Frederic Pincet, and James E. Rothman
FEBS Letters, ISSN: 00145793, eISSN: 18733468, Volume: 593, Pages: 154-162, Published: January 2019 Wiley
The buttressed‐ring hypothesis, supported by recent cryo‐electron tomography analysis of docked synaptic‐like vesicles in neuroendocrine cells, postulates that prefusion SNAREpins are stabilized and organized by Synaptotagmin (Syt) ring‐like oligomers. Here, we use a reconstituted single‐vesicle fusion analysis to test the prediction that destabilizing the Syt1 oligomers destabilizes the clamp and results in spontaneous fusion in the absence of Ca2+. Vesicles in which Syt oligomerization is compromised by a ring‐destabilizing mutation dock and diffuse freely on the bilayer until they fuse spontaneously, similar to vesicles containing only v‐SNAREs. In contrast, vesicles containing wild‐type Syt are immobile as soon as they attach to the bilayer and remain frozen in place, up to at least 1 h until fusion is triggered by Ca2+.
Manindra Bera, Sri Rama Koti Ainavarapu, and Kaushik Sengupta
Scientific Reports, eISSN: 20452322, Published: 15 June 2016 Springer Science and Business Media LLC
Nuclear lamins are type V intermediate filament proteins which form an elastic meshwork underlying the inner nuclear membrane. Lamins directly contribute to maintain the nuclear shape and elasticity. More than 400 mutations have been reported in lamin A that are involved in diseases known as laminopathies. These mutations are scattered mainly in the lamin rod domain along with some in its C-terminal domain. The contribution of the rod domain towards the elasticity of lamin A molecule was hitherto unknown. Here, we have elucidated the significance of the 1B and 2B domains of the rod in modulating the elastic behavior of lamin A by single-molecule force spectroscopy. In addition, we have also studied the network forming capacity of these domains and their corresponding viscoelastic behavior. We have shown that the 1B domain has the ability to form a lamin-like network and resists larger deformation. However at the single-molecular level, both the domains have comparable mechanical properties. The self-assembly of the 1B domain contributes to the elasticity of the lamin A network.
Manindra Bera, Hema Chandra Kotamarthi, Subarna Dutta, Angana Ray, Saptaparni Ghosh, Dhananjay Bhattacharyya, Sri Rama Koti Ainavarapu, and Kaushik Sengupta
Biochemistry, ISSN: 00062960, eISSN: 15204995, Pages: 7247-7258, Published: 25 November 2014 American Chemical Society (ACS)
A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath the nuclear envelope thereby conferring proper shape and mechanical rigidity to the nucleus. Lamin proteins are also shown to be related diversely to basic nuclear processes. More than 400 mutations in human lamin A protein alone have been reported to produce at least 11 different disease conditions jointly termed as laminopathies. These mutations in lamin A are scattered throughout its helical rod domain, as well as the C-terminal domain containing the conserved Ig-fold region. The commonality of phenotypes in all these diseases is characterized by misshapen nuclei of the affected tissues which might stem from altered rigidity of the supporting lamina hence lamins. Here we have focused on autosomal dominant Emery-Dreifuss Muscular Dystrophy, one such laminopathy where R453W is the causative mutation located in the Ig domain of lamin A. We have investigated by single-molecule force spectroscopy how a stretching mechanical perturbation senses the destabilizing effect of the mutation in the lamin A Ig domain and compared the mechanoelastic properties of the mutant R453W with that of the wild-type in conjunction with steered molecular dynamics. Furthermore, we have shown the interaction of Ig domain with emerin, another key player and interacting partner in the pathogenesis of EDMD, is disrupted in the R453W mutant. This altered mechanoresistance of Ig domain itself and consequent uncoupling of lamin A-emerin interaction might underlie the altered mechanotransduction properties of EDMD affected nuclei.