Publications by authors named "Debanjan Goswami"

13 Publications

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Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior.

Elife 2020 Jan 20;9. Epub 2020 Jan 20.

NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, United States.

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.
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http://dx.doi.org/10.7554/eLife.47654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060043PMC
January 2020

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Cancer Res 2020 04 7;80(8):1630-1643. Epub 2020 Jan 7.

National Cancer Institute-RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, Maryland.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185043PMC
April 2020

Deletion of in adult mice impairs basal AMPA receptor transmission and LTP in hippocampal CA1 pyramidal neurons.

Proc Natl Acad Sci U S A 2018 06 21;115(23):E5382-E5389. Epub 2018 May 21.

Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305;

Leucine-rich repeat transmembrane (LRRTM) proteins are synaptic cell adhesion molecules that influence synapse formation and function. They are genetically associated with neuropsychiatric disorders, and via their synaptic actions likely regulate the establishment and function of neural circuits in the mammalian brain. Here, we take advantage of the generation of a and double conditional knockout mouse ( cKO) to examine the role of LRRTM1,2 at mature excitatory synapses in hippocampal CA1 pyramidal neurons. Genetic deletion of in vivo in CA1 neurons using Cre recombinase-expressing lentiviruses dramatically impaired long-term potentiation (LTP), an impairment that was rescued by simultaneous expression of LRRTM2, but not LRRTM4. Mutation or deletion of the intracellular tail of LRRTM2 did not affect its ability to rescue LTP, while point mutations designed to impair its binding to presynaptic neurexins prevented rescue of LTP. In contrast to previous work using shRNA-mediated knockdown of LRRTM1,2, KO of these proteins at mature synapses also caused a decrease in AMPA receptor-mediated, but not NMDA receptor-mediated, synaptic transmission and had no detectable effect on presynaptic function. Imaging of recombinant photoactivatable AMPA receptor subunit GluA1 in the dendritic spines of cultured neurons revealed that it was less stable in the absence of LRRTM1,2. These results illustrate the advantages of conditional genetic deletion experiments for elucidating the function of endogenous synaptic proteins and suggest that LRRTM1,2 proteins help stabilize synaptic AMPA receptors at mature spines during basal synaptic transmission and LTP.
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http://dx.doi.org/10.1073/pnas.1803280115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003336PMC
June 2018

The Retromer Supports AMPA Receptor Trafficking During LTP.

Neuron 2017 Apr;94(1):74-82.e5

Nancy Pritzker Laboratory, Department of Psychiatry & Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305-5453, USA. Electronic address:

Alterations in the function of the retromer, a multisubunit protein complex that plays a specialized role in endosomal sorting, have been linked to Alzheimer's and Parkinson's diseases, yet little is known about the retromer's role in the mature brain. Using in vivo knockdown of the critical retromer component VPS35, we demonstrate a specific role for this endosomal sorting complex in the trafficking of AMPA receptors during NMDA-receptor-dependent LTP at mature hippocampal synapses. The impairment of LTP due to VPS35 knockdown was mechanistically independent of any role of the retromer in the production of Aβ from APP. Finally, we find surprising differences between Alzheimer's- and Parkinson's-disease-linked VPS35 mutations in supporting this pathway. These findings demonstrate a key role for the retromer in LTP and provide insights into how retromer malfunction in the mature brain may contribute to symptoms of common neurodegenerative diseases. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.neuron.2017.03.020DOI Listing
April 2017

Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP.

Nature 2017 04 29;544(7650):316-321. Epub 2017 Mar 29.

Department of Molecular &Cellular Physiology and Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California 94305, USA.

Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca-dependent presynaptic vesicle exocytosis, also blocked Ca-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca-sensors for Ca-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.
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http://dx.doi.org/10.1038/nature21720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734942PMC
April 2017

Modulation of excitation on parvalbumin interneurons by neuroligin-3 regulates the hippocampal network.

Nat Neurosci 2017 02 9;20(2):219-229. Epub 2017 Jan 9.

Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California, USA.

Hippocampal network activity is generated by a complex interplay between excitatory pyramidal cells and inhibitory interneurons. Although much is known about the molecular properties of excitatory synapses on pyramidal cells, comparatively little is known about excitatory synapses on interneurons. Here we show that conditional deletion of the postsynaptic cell adhesion molecule neuroligin-3 in parvalbumin interneurons causes a decrease in NMDA-receptor-mediated postsynaptic currents and an increase in presynaptic glutamate release probability by selectively impairing the inhibition of glutamate release by presynaptic Group III metabotropic glutamate receptors. As a result, the neuroligin-3 deletion altered network activity by reducing gamma oscillations and sharp wave ripples, changes associated with a decrease in extinction of contextual fear memories. These results demonstrate that neuroligin-3 specifies the properties of excitatory synapses on parvalbumin-containing interneurons by a retrograde trans-synaptic mechanism and suggest a molecular pathway whereby neuroligin-3 mutations contribute to neuropsychiatric disorders.
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http://dx.doi.org/10.1038/nn.4471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5272845PMC
February 2017

LTP requires a unique postsynaptic SNARE fusion machinery.

Neuron 2013 Feb;77(3):542-58

Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA.

Membrane fusion during exocytosis is mediated by assemblies of SNARE (soluble NSF-attachment protein receptor) and SM (Sec1/Munc18-like) proteins. The SNARE/SM proteins involved in vesicle fusion during neurotransmitter release are well understood, whereas little is known about the protein machinery that mediates activity-dependent AMPA receptor (AMPAR) exocytosis during long-term potentiation (LTP). Using direct measurements of LTP in acute hippocampal slices and an in vitro LTP model of stimulated AMPAR exocytosis, we demonstrate that the Q-SNARE proteins syntaxin-3 and SNAP-47 are required for regulated AMPAR exocytosis during LTP but not for constitutive basal AMPAR exocytosis. In contrast, the R-SNARE protein synaptobrevin-2/VAMP2 contributes to both regulated and constitutive AMPAR exocytosis. Both the central complexin-binding and the N-terminal Munc18-binding sites of syntaxin-3 are essential for its postsynaptic role in LTP. Thus, postsynaptic exocytosis of AMPARs during LTP is mediated by a unique fusion machinery that is distinct from that used during presynaptic neurotransmitter release.
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http://dx.doi.org/10.1016/j.neuron.2012.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569727PMC
February 2013

Dynamic imaging of homo-FRET in live cells by fluorescence anisotropy microscopy.

Methods Enzymol 2012 ;505:291-327

National Centre for Biological Science, Bangalore, India.

Multiple lipid and protein components of the plasma membrane of a living cell are organized, both compositionally and functionally, at different spatial and temporal scales. For instance, Rab protein domains in membranes the clathrin-coated pit, or the immunological synapse are exquisite examples of functional compartmentalization in cell membranes. These assemblies consist in part of nanoscale complexes of lipids and proteins and are necessary to facilitate some specific sorting and signaling functions. It is evident that cellular functions require a regulated spatiotemporal organization of components at the nanoscale, often comprising of countable number of molecular species. Here, we describe multiple homo-FRET-based imaging methods that provide information about nanoscale interactions between fluorescently tagged molecules in live cells, at optically resolved spatial resolution.
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http://dx.doi.org/10.1016/B978-0-12-388448-0.00024-3DOI Listing
June 2012

Postsynaptic complexin controls AMPA receptor exocytosis during LTP.

Neuron 2012 Jan;73(2):260-7

Nancy Pritzker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 265 Campus Drive, Stanford CA 94305, USA.

Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.
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http://dx.doi.org/10.1016/j.neuron.2011.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269030PMC
January 2012

A DNA nanomachine that maps spatial and temporal pH changes inside living cells.

Nat Nanotechnol 2009 May 6;4(5):325-30. Epub 2009 Apr 6.

National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bellary Road, Bangalore, 560065, India.

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8, with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in sensing, diagnostics and targeted therapies in living systems.
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http://dx.doi.org/10.1038/nnano.2009.83DOI Listing
May 2009

Nanoclusters of GPI-anchored proteins are formed by cortical actin-driven activity.

Cell 2008 Dec;135(6):1085-97

National Centre for Biological Sciences (TIFR), Bellary Road, Bangalore 560065, India.

Several cell-surface lipid-tethered proteins exhibit a concentration-independent, cholesterol-sensitive organization of nanoscale clusters and monomers. To understand the mechanism of formation of these clusters, we investigate the spatial distribution and steady-state dynamics of fluorescently tagged GPI-anchored protein nanoclusters using high-spatial and temporal resolution FRET microscopy. These studies reveal a nonrandom spatial distribution of nanoclusters, concentrated in optically resolvable domains. Monitoring the dynamics of recovery of fluorescence intensity and anisotropy, we find that nanoclusters are immobile, and the dynamics of interconversion between nanoclusters and monomers, over a range of temperatures, is spatially heterogeneous and non-Arrhenius, with a sharp crossover coinciding with a reduction in the activity of cortical actin. Cholesterol depletion perturbs cortical actin and the spatial scale and interconversion dynamics of nanoclusters. Direct perturbations of cortical actin activity also affect the construction, dynamics, and spatial organization of nanoclusters. These results suggest a unique mechanism of complexation of cell-surface molecules regulated by cortical actin activity.
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http://dx.doi.org/10.1016/j.cell.2008.11.032DOI Listing
December 2008

Nanoscale organization of hedgehog is essential for long-range signaling.

Cell 2008 Jun;133(7):1214-27

National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore 560 065, India.

Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.
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http://dx.doi.org/10.1016/j.cell.2008.05.026DOI Listing
June 2008

Precise positioning of myosin VI on endocytic vesicles in vivo.

PLoS Biol 2007 Aug;5(8):e210

Department of Biochemistry, Stanford University Medical Center, Stanford, California, United States of America.

Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET) resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.
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http://dx.doi.org/10.1371/journal.pbio.0050210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1939883PMC
August 2007