Publications by authors named "Avital A Rodal"

37 Publications

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals.

J Cell Biol 2021 Aug 21;220(8). Epub 2021 May 21.

Department of Biology, Brandeis University, Waltham, MA.

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer's disease, and suggest that misregulated EV traffic may be an underlying defect.
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http://dx.doi.org/10.1083/jcb.202012034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144913PMC
August 2021

TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression.

Proc Natl Acad Sci U S A 2020 05 11;117(21):11760-11769. Epub 2020 May 11.

Department of Biology, Brandeis University, Waltham, MA 02453;

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein which forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, altered neuronal dendritic morphology is a common theme among several neurological disorders and is thought to precede neurodegeneration. We previously found that both TDP-43 overexpression (OE) and knockdown (KD) result in reduced dendritic branching of cortical neurons. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as an approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 RNA targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescues defects in dendritic branching. Finally, we demonstrate, using RNA sequencing, that TDP-43 OE and KD cause similar changes in the abundance of specific messenger RNAs, consistent with their ability to produce similar morphological defects. Our data therefore provide a mechanism by which TDP-43 dysfunction interferes with dendritic branching, and may define pathways for therapeutic intervention in neurodegenerative diseases.
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http://dx.doi.org/10.1073/pnas.1917038117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260973PMC
May 2020

Mechanisms for biogenesis and release of neuronal extracellular vesicles.

Curr Opin Neurobiol 2020 08 5;63:104-110. Epub 2020 May 5.

Department of Biology, Brandeis University, 415 South St. MS029, Waltham, MA 02453, United States. Electronic address:

Neurons release membrane-bound extracellular vesicles (EVs) carrying proteins, nucleic acids, and other cargoes to mediate neuronal development, plasticity, inflammation, regeneration, and degeneration. Functional studies and therapeutic interventions into EV-dependent processes will require a deep understanding of how neuronal EVs are formed and released. However, unraveling EV biogenesis and trafficking mechanisms is challenging, since there are multiple pathways governing generation of different types of EVs, which overlap mechanistically with each other, as well as with intracellular endolysosomal trafficking pathways. Further, neurons present special considerations for EVs due to their extreme morphologies and specialization for membrane traffic. Here, we review recent work elucidating neuronal pathways that regulate EV biogenesis and release, with the goal of identifying directed strategies for experimental and therapeutic targeting of specific types of EVs.
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http://dx.doi.org/10.1016/j.conb.2020.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483335PMC
August 2020

The Impact of Sustained Immunization Regimens on the Antibody Response to Oligomannose Glycans.

ACS Chem Biol 2020 03 9;15(3):789-798. Epub 2020 Mar 9.

Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454-9110, United States.

The high mannose patch (HMP) of the HIV envelope protein (Env) is the structure most frequently targeted by broadly neutralizing antibodies; therefore, many researchers have attempted to use mimics of this region as a vaccine immunogen. In our previous efforts, vaccinating rabbits with evolved HMP mimic glycopeptides containing Man resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1→2Man tips of Man glycans. A possible reason could be processing of our immunogen by host serum mannosidases. We sought to test whether more prolonged dosing could increase the antibody response to intact glycans, possibly by increasing the availability of intact Man to germinal centers. Here, we describe a study investigating the impact of immunization regimen on antibody response by testing immunogen delivery through bolus, an exponential series of mini doses, or a continuously infusing mini-osmotic pump. Our results indicate that, with our glycopeptide immunogens, standard bolus immunization elicited the strongest HIV Env-binding antibody response, even though higher overall titers to the glycopeptide were elicited by the exponential and pump regimens. Antibody selectivity for intact glycan was, if anything, slightly better in the bolus-immunized animals.
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http://dx.doi.org/10.1021/acschembio.0c00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7091532PMC
March 2020

Nanoparticles binding to lipid membranes: from vesicle-based gels to vesicle tubulation and destruction.

Nanoscale 2019 Oct;11(39):18464-18474

Department of Physics, University of Massachusetts Amherst, USA.

While cells offer numerous inspiring examples in which membrane morphology and function are controlled by interactions with viruses or proteins, we still lack design principles for controlling membrane morphology in synthetic systems. With experiments and simulations, we show that spherical nanoparticles binding to lipid-bilayer membrane vesicles results in a remarkably rich set of collective morphologies that are controllable via the particle binding energy. We separately study cationic and anionic particles, where the adhesion is tuned by addition of oppositely charged lipids to the vesicles. When the binding energy is weak relative to a characteristic membrane-bending energy, vesicles adhere to one another and form a soft solid gel, a novel and useful platform for controlled release. With larger binding energy, a transition from partial to complete wrapping of the nanoparticles causes a remarkable vesicle destruction process culminating in rupture, nanoparticle-membrane tubules, and an apparent inversion of the vesicles. These findings help unify the diverse phenomena observed previously. They also open the door to a new class of vesicle-based, closed-cell gels that are more than 99% water and can encapsulate and release on demand, and show how to drive intentional membrane remodeling for shape-responsive systems.
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http://dx.doi.org/10.1039/c9nr06570aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155749PMC
October 2019

Higher-order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes.

J Cell Biol 2019 08 28;218(8):2600-2618. Epub 2019 Jun 28.

Department of Biology, Brandeis University, Waltham, MA

The activities of neuronal signaling receptors depend heavily on the maturation state of the endosomal compartments in which they reside. However, it remains unclear how the distribution of these compartments within the uniquely complex morphology of neurons is regulated and how this distribution itself affects signaling. Here, we identified mechanisms by which Sorting Nexin 16 (SNX16) controls neuronal endosomal maturation and distribution. We found that higher-order assembly of SNX16 via its coiled-coil (CC) domain drives membrane tubulation in vitro and endosome association in cells. In motor neurons, activation of Rab5 and CC-dependent self-association of SNX16 lead to its endosomal enrichment, accumulation in Rab5- and Rab7-positive tubulated compartments in the cell body, and concomitant depletion of SNX16-positive endosomes from the synapse. This results in accumulation of synaptic growth-promoting bone morphogenetic protein receptors in the cell body and correlates with increased synaptic growth. Our results indicate that Rab regulation of SNX16 assembly controls the endosomal distribution and signaling activities of receptors in neurons.
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http://dx.doi.org/10.1083/jcb.201811074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683739PMC
August 2019

The enemy of my enemy: PTEN and PLCXD collude to fight endosomal PtdIns(4,5)P.

J Cell Biol 2019 Jul 12;218(7):2082-2083. Epub 2019 Jun 12.

Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA

Loss of the phosphoinositide 5-phosphatase OCRL causes accumulation of PtdIns(4,5)P on membranes and, ultimately, Lowe syndrome. In this issue, Mondin et al. (2019. https://doi.org/10.1083/jcb.201805155) discover that a surprising partnership between PTEN and the phospholipase PLCXD can compensate for OCRL to suppress endosomal PtdIns(4,5)P accumulation.
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http://dx.doi.org/10.1083/jcb.201906022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605809PMC
July 2019

Enzymatic Assemblies Disrupt the Membrane and Target Endoplasmic Reticulum for Selective Cancer Cell Death.

J Am Chem Soc 2018 08 24;140(30):9566-9573. Epub 2018 Jul 24.

Department of Chemistry , Brandeis University , 415 South Street , Waltham , Massachusetts 02454 , United States.

The endoplasmic reticulum (ER) is responsible for the synthesis and folding of a large number of proteins, as well as intracellular calcium regulation, lipid synthesis, and lipid transfer to other organelles, and is emerging as a target for cancer therapy. However, strategies for selectively targeting the ER of cancer cells are limited. Here we show that enzymatically generated crescent-shaped supramolecular assemblies of short peptides disrupt cell membranes and target ER for selective cancer cell death. As revealed by sedimentation assay, the assemblies interact with synthetic lipid membranes. Live cell imaging confirms that the assemblies impair membrane integrity, which is further supported by lactate dehydrogenase (LDH) assays. According to transmission electron microscopy (TEM), static light scattering (SLS), and critical micelle concentration (CMC), attaching an l-amino acid at the C-terminal of a d-tripeptide results in the crescent-shaped supramolecular assemblies. Structure-activity relationship suggests that the crescent-shaped morphology is critical for interacting with membranes and for controlling cell fate. Moreover, fluorescent imaging indicates that the assemblies accumulate on the ER. Time-dependent Western blot and ELISA indicate that the accumulation causes ER stress and subsequently activates the caspase signaling cascade for cell death. As an approach for in situ generating membrane binding scaffolds (i.e., the crescent-shaped supramolecular assemblies), this work promises a new way to disrupt the membrane and to target the ER for developing anticancer therapeutics.
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http://dx.doi.org/10.1021/jacs.8b04641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070399PMC
August 2018

Active Probes for Imaging Membrane Dynamics of Live Cells with High Spatial and Temporal Resolution over Extended Time Scales and Areas.

J Am Chem Soc 2018 03 1;140(10):3505-3509. Epub 2018 Mar 1.

Department of Chemistry , Brandeis University , 415 South Street , Waltham , Massachusetts 02453 , United States.

Despite the advancement of molecular imaging techniques, there is an unmet need for probes for direct imaging of membrane dynamics of live cells. Here we report a novel type of active (or enzyme responsive) probes to directly image membrane dynamics of live cells with high spatial and temporal resolution over extended time scales and areas. Because lipid rafts enrich cholesterols and GPI-anchored enzymes (e.g., ectophosphatases), we design probes that consist of an enzymatic trigger, a fluorophore, and a cholesterol that are affinitive to the cell membrane. Being water-soluble and as the substrate of ectophosphatase, these cell compatible probes preferentially and rapidly assemble in plasma membrane, exhibit strong fluorescence, work at micromolar concentrations, and easily achieve high resolution monitoring of nanoscale heterogeneity in membranes of live cells, the release of exosomes, and the membrane dynamics of live cells. This work provides a facile means to link membrane dynamics and heterogeneity to cellular processes for understanding the interactions between membranes and proteins.
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http://dx.doi.org/10.1021/jacs.7b13307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858877PMC
March 2018

Cellular Uptake of A Taurine-Modified, Ester Bond-Decorated D-Peptide Derivative via Dynamin-Based Endocytosis and Macropinocytosis.

Mol Ther 2018 02 6;26(2):648-658. Epub 2018 Jan 6.

Department of Chemistry, Brandeis University, 415 South St., Waltham, MA 02453, USA. Electronic address:

Most of the peptides used for promoting cellular uptake bear positive charges. In our previous study, we reported an example of taurine (bearing negative charges in physiological conditions) promoting cellular uptake of D-peptides. Taurine, conjugated to a small D-peptide via an ester bond, promotes the cellular uptake of this D-peptide. Particularly, intracellular carboxylesterase (CES) instructs the D-peptide to self-assemble and to form nanofibers, which largely disfavors efflux and further enhances the intracellular accumulation of the D-peptide, as supported by that the addition of CES inhibitors partially impaired cellular uptake of this molecule in mammalian cell lines. Using dynamin 1, 2, and 3 triple knockout (TKO) mouse fibroblasts, we demonstrated that cells took up this molecule via macropinocytosis and dynamin-dependent endocytosis. Imaging of Drosophila larval blood cells derived from endocytic mutants confirmed the involvement of multiple endocytosis pathways. Electron microscopy (EM) indicated that the precursors can form aggregates on the cell surface to facilitate the cellular uptake via macropinocytosis. EM also revealed significantly increased numbers of vesicles in the cytosol. This work provides new insights into the cellular uptake of taurine derivative for intracellular delivery and self-assembly of D-peptides.
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http://dx.doi.org/10.1016/j.ymthe.2017.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835119PMC
February 2018

TDP-43 misexpression causes defects in dendritic growth.

Sci Rep 2017 Nov 15;7(1):15656. Epub 2017 Nov 15.

Department of Biology, Volen Center for Complex Systems, and National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts, 02454, USA.

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) share overlapping genetic causes and disease symptoms, and are linked neuropathologically by the RNA binding protein TDP-43 (TAR DNA binding protein-43 kDa). TDP-43 regulates RNA metabolism, trafficking, and localization of thousands of target genes. However, the cellular and molecular mechanisms by which dysfunction of TDP-43 contributes to disease pathogenesis and progression remain unclear. Severe changes in the structure of neuronal dendritic arbors disrupt proper circuit connectivity, which in turn could contribute to neurodegenerative disease. Although aberrant dendritic morphology has been reported in non-TDP-43 mouse models of ALS and in human ALS patients, this phenotype is largely unexplored with regards to TDP-43. Here we have employed a primary rodent neuronal culture model to study the cellular effects of TDP-43 dysfunction in hippocampal and cortical neurons. We show that manipulation of TDP-43 expression levels causes significant defects in dendritic branching and outgrowth, without an immediate effect on cell viability. The effect on dendritic morphology is dependent on the RNA-binding ability of TDP-43. Thus, this model system will be useful in identifying pathways downstream of TDP-43 that mediate dendritic arborization, which may provide potential new avenues for therapeutic intervention in ALS/FTD.
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http://dx.doi.org/10.1038/s41598-017-15914-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688077PMC
November 2017

An in situ Dynamic Continuum of Supramolecular Phosphoglycopeptides Enables Formation of 3D Cell Spheroids.

Angew Chem Int Ed Engl 2017 12 22;56(51):16297-16301. Epub 2017 Nov 22.

Department of chemistry, Brandeis University, 415 South St, Waltham, MA, 02454, USA.

Higher-order assemblies of proteins, with a structural and dynamic continuum, is an important concept in biology, but these insights have yet to be applied in designing biomaterials. Dynamic assemblies of supramolecular phosphoglycopeptides (sPGPs) transform a 2D cell sheet into 3D cell spheroids. A ligand-receptor interaction between a glycopeptide and a phosphopeptide produces sPGPs that form nanoparticles, which transform into nanofibrils upon partial enzymatic dephosphorylation. The assemblies form dynamically and hierarchically in situ on the cell surface, and interact with the extracellular matrix molecules and effectively abolish contact inhibition of locomotion (CIL) of the cells. Integrating molecular recognition, catalysis, and assembly, these active assemblies act as a dynamic continuum to disrupt CIL, thus illustrating a new kind of biomaterial for regulating cell behavior.
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http://dx.doi.org/10.1002/anie.201710269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857944PMC
December 2017

dOCRL maintains immune cell quiescence by regulating endosomal traffic.

PLoS Genet 2017 Oct 13;13(10):e1007052. Epub 2017 Oct 13.

Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, Massachusetts, United States of America.

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.
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http://dx.doi.org/10.1371/journal.pgen.1007052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656325PMC
October 2017

The membrane strikes back: phosphoinositide binding regulates Skywalker function.

Nat Struct Mol Biol 2016 11;23(11):956-957

Department of Biology, Brandeis University, Waltham, Massachusetts, USA.

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http://dx.doi.org/10.1038/nsmb.3313DOI Listing
November 2016

Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck.

Proc Natl Acad Sci U S A 2016 09 6;113(38):E5552-61. Epub 2016 Sep 6.

Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, MA 02453

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035868PMC
http://dx.doi.org/10.1073/pnas.1524412113DOI Listing
September 2016

Beyond the SNARE: Munc18-1 chaperones α-synuclein.

J Cell Biol 2016 09 5;214(6):641-3. Epub 2016 Sep 5.

Department of Biology, Brandeis University, Waltham, MA 02453

Early infantile epileptic encephalopathy (EIEE)-associated mutations in MUNC18-1 cause Munc18-1 misfolding and cellular aggregation. In this issue, Chai et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201512016) find that Munc18-1 is a molecular chaperone for α-synuclein and that aggregated Munc18-1 EIEE-causing mutants promote α-synuclein aggregation.
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http://dx.doi.org/10.1083/jcb.201608060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021099PMC
September 2016

Role of BMP receptor traffic in synaptic growth defects in an ALS model.

Mol Biol Cell 2016 10 17;27(19):2898-910. Epub 2016 Aug 17.

Department of Biology, Brandeis University, Waltham, MA 02453

TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to amyotrophic lateral sclerosis (ALS) and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is believed to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it is unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. Here we found that both loss and gain of function of TDP-43 in Drosophila cause a reduction of synaptic growth-promoting bone morphogenic protein (BMP) signaling at the neuromuscular junction (NMJ). Further, we observed a shift of BMP receptors from early to recycling endosomes and increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution and suppressed BMP signaling, synaptic growth, and larval crawling defects. Our results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042577PMC
http://dx.doi.org/10.1091/mbc.E16-07-0519DOI Listing
October 2016

Tissue-specific tagging of endogenous loci in Drosophila melanogaster.

Biol Open 2015 Dec 23;5(1):83-9. Epub 2015 Dec 23.

Department of Biology, Brandeis University, 415 South St, Waltham, MA 02454, USA

Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins) that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues.
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http://dx.doi.org/10.1242/bio.016089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728311PMC
December 2015

Membrane Charge Directs the Outcome of F-BAR Domain Lipid Binding and Autoregulation.

Cell Rep 2015 Dec 10;13(11):2597-2609. Epub 2015 Dec 10.

Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, MA 02453, USA. Electronic address:

F-BAR domain proteins regulate and sense membrane curvature by interacting with negatively charged phospholipids and assembling into higher-order scaffolds. However, regulatory mechanisms controlling these interactions are poorly understood. Here, we show that Drosophila Nervous Wreck (Nwk) is autoregulated by a C-terminal SH3 domain module that interacts directly with its F-BAR domain. Surprisingly, this autoregulation does not mediate a simple "on-off" switch for membrane remodeling. Instead, the isolated Nwk F-BAR domain efficiently assembles into higher-order structures and deforms membranes only within a limited range of negative membrane charge, and autoregulation elevates this range. Thus, autoregulation could either reduce membrane binding or promote higher-order assembly, depending on local cellular membrane composition. Our findings uncover an unexpected mechanism by which lipid composition directs membrane remodeling.
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http://dx.doi.org/10.1016/j.celrep.2015.11.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790443PMC
December 2015

The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila.

Traffic 2016 Feb 9;17(2):87-101. Epub 2015 Dec 9.

Department of Biology, Brandeis University, Waltham, MA, USA.

Neurons require target-derived autocrine and paracrine growth factors to maintain proper identity, innervation, homeostasis and survival. Neuronal growth factor signaling is highly dependent on membrane traffic, both for the packaging and release of the growth factors themselves, and for regulation of intracellular signaling by their transmembrane receptors. Here, we review recent findings from the Drosophila larval neuromuscular junction (NMJ) that illustrate how specific steps of intracellular traffic and inter-organelle interactions impinge on signaling, particularly in the bone morphogenic protein, Wingless and c-Jun-activated kinase pathways, regulating elaboration and stability of NMJ arbors, construction of synapses and synaptic transmission and homeostasis. These membrane trafficking and signaling pathways have been implicated in human motor neuron diseases including amyotrophic lateral sclerosis and hereditary spastic paraplegia, highlighting their importance for neuronal health and survival.
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http://dx.doi.org/10.1111/tra.12345DOI Listing
February 2016

Assembly of actin filaments and microtubules in Nwk F-BAR-induced membrane deformations.

Commun Integr Biol 2015 Mar-Apr;8(2):e1000703. Epub 2015 Apr 29.

Department of Biology; Brandeis University ; Waltham, MA USA.

F-BAR domains form crescent-shaped dimers that bind to and deform lipid bilayers, and play a role in many cellular processes requiring membrane remodeling, including endocytosis and cell morphogenesis. Nervous Wreck (NWK) encodes an F-BAR/SH3 protein that regulates synapse growth in Drosophila. Unlike conventional F-BAR proteins that assemble tip-to-tip into filaments and helical arrays around membrane tubules, the Nwk F-BAR domain instead assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. In cells, this membrane deforming activity generates small buds, which can lengthen into extensive protrusions upon actin cytoskeleton polymerization. Here, we show that Nwk-induced cellular protrusions contain dynamic microtubules, distinguishing them from conventional filopodia, and further do not depend on actin filaments or microtubules for their maintenance. Our results indicate new ways in which close cooperation between the membrane remodeling and cytoskeletal machinery underlies large-scale changes in cellular morphology.
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http://dx.doi.org/10.1080/19420889.2014.1000703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594231PMC
October 2015

The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function.

Mol Biol Cell 2015 Sep 22;26(18):3275-88. Epub 2015 Jul 22.

Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, MA 02453

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. Here, we show that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. Our results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR and uncover new roles for an EHD protein at synapses.
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http://dx.doi.org/10.1091/mbc.E15-02-0093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569317PMC
September 2015

Drosophila comes of age as a model system for understanding the function of cytoskeletal proteins in cells, tissues, and organisms.

Cytoskeleton (Hoboken) 2015 May 30;72(5):207-24. Epub 2015 Jun 30.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts.

For the last 100 years, Drosophila melanogaster has been a powerhouse genetic system for understanding mechanisms of inheritance, development, and behavior in animals. In recent years, advances in imaging and genetic tools have led to Drosophila becoming one of the most effective systems for unlocking the subcellular functions of proteins (and particularly cytoskeletal proteins) in complex developmental settings. In this review, written for non-Drosophila experts, we will discuss critical technical advances that have enabled these cell biological insights, highlighting three examples of cytoskeletal discoveries that have arisen as a result: (1) regulation of Arp2/3 complex in myoblast fusion, (2) cooperation of the actin filament nucleators Spire and Cappuccino in establishment of oocyte polarity, and (3) coordination of supracellular myosin cables. These specific examples illustrate the unique power of Drosophila both to uncover new cytoskeletal structures and functions, and to place these discoveries in a broader in vivo context, providing insights that would have been impossible in a cell culture model or in vitro. Many of the cellular structures identified in Drosophila have clear counterparts in mammalian cells and tissues, and therefore elucidating cytoskeletal functions in Drosophila will be broadly applicable to other organisms.
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http://dx.doi.org/10.1002/cm.21228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4782189PMC
May 2015

New approaches for studying synaptic development, function, and plasticity using Drosophila as a model system.

J Neurosci 2013 Nov;33(45):17560-8

Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, Stanford Institute for Neuro-innovation and Translational Neurosciences and Department of Neurosurgery, Stanford University School of Medicine, Palo Alto, California 94305, Department of Molecular Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, Department of Biology, Brandeis University, Waltham, Massachusetts 02453, National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, Maryland 20892, VIB, Center for the Biology of Disease and KU Leuven, Department for Human Genetics, 3000 Leuven, Belgium, and Department of Biology, University of Southern California, Los Angeles, California 90089.

The fruit fly Drosophila melanogaster has been established as a premier experimental model system for neuroscience research. These organisms are genetically tractable, yet their nervous systems are sufficiently complex to study diverse processes that are conserved across metazoans, including neural cell fate determination and migration, axon guidance, synaptogenesis and function, behavioral neurogenetics, and responses to neuronal injury. For several decades, Drosophila neuroscientists have taken advantage of a vast toolkit of genetic and molecular techniques to reveal fundamental principles of neuroscience illuminating to all systems, including the first behavioral mutants from Seymour Benzer's pioneering work in the 1960s and 1970s, the cloning of the first potassium channel in the 1980s, and the identification of the core genes that orchestrate axon guidance and circadian rhythms in the 1990s. Over the past decade, new tools and innovations in genetic, imaging, and electrophysiological technologies have enabled the visualization, in vivo, of dynamic processes in synapses with unprecedented resolution. We will review some of the fresh insights into synaptic development, function, and plasticity that have recently emerged in Drosophila with an emphasis on the unique advantages of this model system.
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http://dx.doi.org/10.1523/JNEUROSCI.3261-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818537PMC
November 2013

Formation of membrane ridges and scallops by the F-BAR protein Nervous Wreck.

Mol Biol Cell 2013 Aug 12;24(15):2406-18. Epub 2013 Jun 12.

Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, MA 02454, USA.

Eukaryotic cells are defined by extensive intracellular compartmentalization, which requires dynamic membrane remodeling. FER/Cip4 homology-Bin/amphiphysin/Rvs (F-BAR) domain family proteins form crescent-shaped dimers, which can bend membranes into buds and tubules of defined geometry and lipid composition. However, these proteins exhibit an unexplained wide diversity of membrane-deforming activities in vitro and functions in vivo. We find that the F-BAR domain of the neuronal protein Nervous Wreck (Nwk) has a novel higher-order structure and membrane-deforming activity that distinguishes it from previously described F-BAR proteins. The Nwk F-BAR domain assembles into zigzags, creating ridges and periodic scallops on membranes in vitro. This activity depends on structural determinants at the tips of the F-BAR dimer and on electrostatic interactions of the membrane with the F-BAR concave surface. In cells, Nwk-induced scallops can be extended by cytoskeletal forces to produce protrusions at the plasma membrane. Our results define a new F-BAR membrane-deforming activity and illustrate a molecular mechanism by which positively curved F-BAR domains can produce a variety of membrane curvatures. These findings expand the repertoire of F-BAR domain mediated membrane deformation and suggest that unique modes of higher-order assembly can define how these proteins sculpt the membrane.
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http://dx.doi.org/10.1091/mbc.E13-05-0271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3727933PMC
August 2013

Drosophila cyfip regulates synaptic development and endocytosis by suppressing filamentous actin assembly.

PLoS Genet 2013 Apr 4;9(4):e1003450. Epub 2013 Apr 4.

Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.
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http://dx.doi.org/10.1371/journal.pgen.1003450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616907PMC
April 2013

Structure and function of the interacting domains of Spire and Fmn-family formins.

Proc Natl Acad Sci U S A 2011 Jul 5;108(29):11884-9. Epub 2011 Jul 5.

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.
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http://dx.doi.org/10.1073/pnas.1105703108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141961PMC
July 2011

A presynaptic endosomal trafficking pathway controls synaptic growth signaling.

J Cell Biol 2011 Apr;193(1):201-17

Department of Biology, The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)-mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments.
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http://dx.doi.org/10.1083/jcb.201009052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082179PMC
April 2011

Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth.

J Neurosci 2008 Aug;28(33):8316-25

The Picower Institute for Learning and Memory, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Regulation of synaptic morphology depends on endocytosis of activated growth signal receptors, but the mechanisms regulating this membrane-trafficking event are unclear. Actin polymerization mediated by Wiskott-Aldrich syndrome protein (WASp) and the actin-related protein 2/3 complex generates forces at multiple stages of endocytosis. FCH-BIN amphiphysin RVS (F-BAR)/SH3 domain proteins play key roles in this process by coordinating membrane deformation with WASp-dependent actin polymerization. However, it is not known how other WASp ligands, such as the small GTPase Cdc42, coordinate with F-BAR/SH3 proteins to regulate actin polymerization at membranes. Nervous Wreck (Nwk) is a conserved neuronal F-BAR/SH3 protein that localizes to periactive zones at the Drosophila larval neuromuscular junction (NMJ) and is required for regulation of synaptic growth via bone morphogenic protein signaling. Here, we show that Nwk interacts with the endocytic proteins dynamin and Dap160 and functions together with Cdc42 to promote WASp-mediated actin polymerization in vitro and to regulate synaptic growth in vivo. Cdc42 function is associated with Rab11-dependent recycling endosomes, and we show that Rab11 colocalizes with Nwk at the NMJ. Together, our results suggest that synaptic growth activated by growth factor signaling is controlled at an endosomal compartment via coordinated Nwk and Cdc42-dependent actin assembly.
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http://dx.doi.org/10.1523/JNEUROSCI.2304-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2546611PMC
August 2008

Synaptic endocytosis: illuminating the role of clathrin assembly.

Curr Biol 2008 Mar;18(6):R259-61

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Acute photo-inactivation of clathrin in Drosophila synapses sheds new light on a 35-year-old debate over mechanisms of synaptic-vesicle endocytosis: clathrin is essential for reformation of functional synaptic vesicles, but not for bulk membrane internalization.
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http://dx.doi.org/10.1016/j.cub.2008.02.014DOI Listing
March 2008
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