Gladfelter Amy S.

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Amy S.

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Now showing 1 - 13 of 13
  • Article
    The hierarchical assembly of septins revealed by high-speed AFM
    (Nature Research, 2020-10-08) Jiao, Fang ; Cannon, Kevin S. ; Lin, Yi-Chih ; Gladfelter, Amy S. ; Scheuring, Simon
    Septins are GTP-binding proteins involved in diverse cellular processes including division and membrane remodeling. Septins form linear, palindromic heteromeric complexes that can assemble in filaments and higher-order structures. Structural studies revealed various septin architectures, but questions concerning assembly-dynamics and -pathways persist. Here we used high-speed atomic force microscopy (HS-AFM) and kinetic modeling which allowed us to determine that septin filament assembly was a diffusion-driven process, while formation of higher-order structures was complex and involved self-templating. Slightly acidic pH and increased monovalent ion concentrations favor filament-assembly, -alignment and -pairing. Filament-alignment and -pairing further favored diffusion-driven assembly. Pairing is mediated by the septin N-termini face, and may occur symmetrically or staggered, likely important for the formation of higher-order structures of different shapes. Multilayered structures are templated by the morphology of the underlying layers. The septin C-termini face, namely the C-terminal extension of Cdc12, may be involved in membrane binding.
  • Article
    Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton
    (Rockefeller University Press, 2016-04-04) Bridges, Andrew A. ; Jentzsch, Maximilian S. ; Oakes, Patrick W. ; Occhipinti, Patricia ; Gladfelter, Amy S.
    Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape.
  • Article
    How nontraditional model systems can save us
    (American Society for Cell Biology, 2015-11-01) Gladfelter, Amy S.
    In this essay I would like to highlight how work in nontraditional model systems is an imperative for our society to prepare for problems we do not even know exist. I present examples of how discovery in nontraditional systems has been critical for fundamental advancement in cell biology. I also discuss how as a collective we might harvest both new questions and new solutions to old problems from the underexplored reservoir of diversity in the biosphere. With advancements in genomics, proteomics, and genome editing, it is now technically feasible for even a single research group to introduce a new model system. I aim here to inspire people to think beyond their familiar model systems and to press funding agencies to support the establishment of new model systems.
  • Article
    Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling
    (American Society for Cell Biology, 2021-09-09) Woods, Benjamin L. ; Cannon, Kevin S. ; Vogt, Ellysa J. D. ; Crutchley, John M. ; Gladfelter, Amy S.
    The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the nonessential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain (cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled.
  • Article
    FXR1 splicing is important for muscle development and biomolecular condensates in muscle cells
    (Rockefeller University Press, 2020-03-13) Smith, Jean A. ; Curry, Ennessa G. ; Blue, R. Eric ; Roden, Christine ; Dundon, Samantha E.R. ; Rodríguez-Vargas, Anthony ; Jordan, Danielle C. ; Chen, Xiaomin ; Lyons, Shawn M. ; Crutchley, John M. ; Anderson, Paul ; Horb, Marko E. ; Gladfelter, Amy S. ; Giudice, Jimena
    Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.
  • Preprint
    mRNA structure determines specificity of a polyQ-driven phase separation
    ( 2018-04) Langdon, Erin M. ; Qiu, Yupeng ; Ghanbari Niaki, Amirhossein ; McLaughlin, Grace A. ; Weidmann, Chase ; Gerbich, Therese M. ; Smith, Jean A. ; Crutchley, John M. ; Termini, Christina M. ; Weeks, Kevin M. ; Myong, Sua ; Gladfelter, Amy S.
    RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.
  • Article
    Fungi in the Marine Environment: Open Questions and Unsolved Problems
    (American Society for Microbiology, 2019-03-05) Amend, Anthony ; Burgaud, Gaëtan ; Cunliffe, Michael ; Edgcomb, Virginia P. ; Ettinger, Cassandra L. ; Gutiérrez, M. H. ; Heitman, Joseph ; Hom, Erik F. Y. ; Ianiri, Giuseppe ; Jones, Adam C. ; Kagami, Maiko ; Picard, Kathryn T. ; Quandt, C. Alisha ; Raghukumar, Seshagiri ; Riquelme, Mertixell ; Stajich, Jason ; Vargas-Muñiz, José ; Walker, Allison K. ; Yarden, Oded ; Gladfelter, Amy S.
    Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.
  • Article
    Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals
    (Rockefeller University Press, 2011-06-13) DeMay, Bradley S. ; Bai, Xiaobo ; Howard, Louisa ; Occhipinti, Patricia ; Meseroll, Rebecca A. ; Spiliotis, Elias T. ; Oldenbourg, Rudolf ; Gladfelter, Amy S.
    The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.
  • Article
    Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking
    (American Society for Cell Biology, 2020-06-29) McLaughlin, Grace A. ; Langdon, Erin M. ; Crutchley, John M. ; Holt, Liam J. ; Forest, M. Gregory ; Newby, Jay M. ; Gladfelter, Amy S.
    The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm). We developed an image analysis pipeline for 3D imaging of GEMs in the context of large, multinucleate fungi where there is evidence of functional compartmentalization of the cytosol for both the nuclear division cycle and branching. We applied a neural network to track particles in 3D and then created quantitative visualizations of spatially varying diffusivity. Using this pipeline to analyze spatial diffusivity patterns, we found that there is substantial variability in the properties of the cytosol. We detected zones where GEMs display especially low diffusivity at hyphal tips and near some nuclei, showing that the physical state of the cytosol varies spatially within a single cell. Additionally, we observed significant cell-to-cell variability in the average diffusivity of GEMs. Thus, the physical properties of the cytosol vary substantially in time and space and can be a source of heterogeneity within individual cells and across populations.
  • Article
    Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium
    (American Society for Cell Biology, 2016-05-18) Dundon, Samantha E.R. ; Chang, Shyr-Shea ; Kumar, Abhishek ; Occhipinti, Patricia ; Shroff, Hari ; Roper, Marcus ; Gladfelter, Amy S.
    Nuclei in syncytia found in fungi, muscles, and tumors can behave independently despite cytoplasmic translation and the homogenizing potential of diffusion. We use a dynactin mutant strain of the multinucleate fungus Ashbya gossypii with highly clustered nuclei to assess the relative contributions of nucleus and cytoplasm to nuclear autonomy. Remarkably, clustered nuclei maintain cell cycle and transcriptional autonomy; therefore some sources of nuclear independence function even with minimal cytosol insulating nuclei. In both nuclear clusters and among evenly spaced nuclei, a nucleus’ transcriptional activity dictates local cytoplasmic contents, as assessed by the localization of several cyclin mRNAs. Thus nuclear activity is a central determinant of the local cytoplasm in syncytia. Of note, we found that the number of nuclei per unit cytoplasm was identical in the mutant to that in wild-type cells, despite clustered nuclei. This work demonstrates that nuclei maintain autonomy at a submicrometer scale and simultaneously maintain a normal nucleocytoplasmic ratio across a syncytium up to the centimeter scale.
  • Preprint
    Septin assemblies form by diffusion-driven annealing on membranes
    ( 2013-12) Bridges, Andrew A. ; Zhang, Huaiying ; Mehta, Shalin B. ; Occhipinti, Patricia ; Tani, Tomomi ; Gladfelter, Amy S.
    Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knit together into higher-order assemblies. Using fluorescence correlation spectroscopy (FCS), we determined that cytosolic septins are in small complexes suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence (TIRF) microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends and do not exchange subunits in the middle of filaments. This work shows for the first time that annealing is an intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.
  • Article
    Phosphoregulation provides specificity to biomolecular condensates in the cell cycle and cell polarity
    (Rockefeller University Press, 2020-07-06) Gerbich, Therese M. ; McLaughlin, Grace A. ; Cassidy, Katelyn ; Gerber, Scott ; Adalsteinsson, David ; Gladfelter, Amy S.
    Biomolecular condensation is a way of organizing cytosol in which proteins and nucleic acids coassemble into compartments. In the multinucleate filamentous fungus Ashbya gossypii, the RNA-binding protein Whi3 regulates the cell cycle and cell polarity through forming macromolecular structures that behave like condensates. Whi3 has distinct spatial localizations and mRNA targets, making it a powerful model for how, when, and where specific identities are established for condensates. We identified residues on Whi3 that are differentially phosphorylated under specific conditions and generated mutants that ablate this regulation. This yielded separation of function alleles that were functional for either cell polarity or nuclear cycling but not both. This study shows that phosphorylation of individual residues on molecules in biomolecular condensates can provide specificity that gives rise to distinct functional identities in the same cell.
  • Article
    An amphipathic helix enables septins to sense micrometer-scale membrane curvature
    (Rockefeller University Press, 2019-01-18) Cannon, Kevin S. ; Woods, Benjamin L. ; Crutchley, John M. ; Gladfelter, Amy S.
    Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.