Cellular Dynamics Program
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The Cellular Dynamics Program (CDP) at the MBL aims to accelerate the knowledge of basic biology and disease through the development and application of biophysical methods customized to shed light on life’s most essential processes.
CDP is composed of several independent laboratories, notably those with a specialization in imaging and cell physiology and biochemistry. Additionally the program houses the NIH:NCRR national resource, the BioCurrents Research Center. The CDP imaging component focuses on the architectural dynamics of living cells, which encompass the timely and coordinated assembly and disassembly of macromolecular structures essential for the proper functioning and differentiation of cells, the spatial and temporal organization of these structures, and their physiological and genetic control. The molecular physiology component, in pursuing studies of cell metabolism and transport biophysics, has pioneered the use of electrochemical sensors to define the chemical signatures surrounding living cells and tissues, opening insights to cell function from a distance.
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PreprintAchiral symmetry breaking and positive Gaussian modulus lead to scalloped colloidal membranes( 2016-11) Gibaud, Thomas ; Kaplan, C. Nadir ; Sharma, Prerna ; Ward, Andrew ; Zakhary, Mark J. ; Oldenbourg, Rudolf ; Meyer, Robert B. ; Kamien, Randall D. ; Powers, Thomas R. ; Dogic, ZvonimirIn the presence of a non-adsorbing polymer, monodisperse rod-like particles assemble into colloidal membranes, which are one rod-length thick liquid-like monolayers of aligned rods. Unlike 3D edgeless bilayer vesicles, colloidal monolayer membranes form open structures with an exposed edge, thus presenting an opportunity to study physics of thin elastic sheets. Membranes assembled from single-component chiral rods form flat disks with uniform edge twist. In comparison, membranes comprised of mixture of rods with opposite chiralities can have the edge twist of either handedness. In this limit disk-shaped membranes become unstable, instead forming structures with scalloped edges, where two adjacent lobes with opposite handedness are separated by a cusp-shaped point defect. Such membranes adopt a 3D configuration, with cusp defects alternatively located above and below the membrane plane. In the achiral regime the cusp defects have repulsive interactions, but away from this limit we measure effective long-ranged attractive binding. A phenomenological model shows that the increase in the edge energy of scalloped membranes is compensated by concomitant decrease in the deformation energy due to Gaussian curvature associated with scalloped edges, demonstrating that colloidal membranes have positive Gaussian modulus. A simple excluded volume argument predicts the sign and magnitude of the Gaussian curvature modulus that is in agreement with experimental measurements. Our results provide insight into how the interplay between membrane elasticity, geometrical frustration and achiral symmetry breaking can be used to fold colloidal membranes into 3D shapes.
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ArticleAn 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.
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ArticleArp2/3 complex inhibition radically alters lamellipodial actin architecture, suspended cell shape, and the cell spreading process(American Society for Cell Biology, 2015-01-07) Henson, John H. ; Yeterian, Mesrob ; Weeks, Richard M. ; Medrano, Angela E. ; Brown, Briana L. ; Geist, Heather L. ; Pais, Mollyann D. ; Oldenbourg, Rudolf ; Shuster, Charles B.Recent studies have investigated the dendritic actin cytoskeleton of the cell edge's lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.
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ArticleBipolarization and poleward flux correlate during xenopus extract spindle assembly(American Society for Cell Biology, 2004-09-22) Mitchison, Timothy J. ; Maddox, P. ; Groen, Aaron C. ; Cameron, Lisa ; Perlman, Z. ; Ohi, Ryoma ; Desai, Ankur R. ; Salmon, Edward D. ; Kapoor, Tarun M.We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.
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ArticleCalyculin-A induces cleavage in a random plane in unfertilized sea urchin eggs(Marine Biological Laboratory, 2009-02) Goda, Makoto ; Inoue, Shinya ; Mabuchi, IsseiCalyculin-A (CLA), a protein phosphatase inhibitor, has been known to induce cleavage resembling normal furrowing in unfertilized sea urchin eggs. In CLA-treated eggs, actin filaments and myosin assemble to form a contractile ring-like structure in the egg cortex; however, this occurs in the absence of a mitotic spindle or asters. Here, we investigated the relationship between the plane of CLA-induced cleavage and the intrinsic animal-vegetal polar axis in sea urchin eggs. The animal-vegetal axis was established using black ink to visualize the jelly canal located at the animal pole in the jelly coat surrounding the egg. We measured the acute angle between the jelly canal axis and the cleavage plane for both fertilized eggs and CLA-treated unfertilized eggs. Although the acute angle lay within 10 degrees for most of the fertilized eggs, it varied widely for CLA-treated unfertilized eggs. Measurements of the diameter of blastomeres revealed that cleavage of fertilized eggs took place in the mid-plane of the egg, but that CLA-induced divisions were unequal. These results suggest that neither the orientation nor the location of the CLA-induced cleavage furrow is related to the animal-vegetal polar axis of the egg, even though the furrowing mechanism itself is not dissimilar to that in fertilized eggs.
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ArticleChromosome malorientations after meiosis II arrest cause nondisjunction(American Society for Cell Biology, 2007-02-21) Janicke, Marie A. ; Lasko, Loren ; Oldenbourg, Rudolf ; LaFountain, James R.This study investigated the basis of meiosis II nondisjunction. Cold arrest induced a fraction of meiosis II crane fly spermatocytes to form (n + 1) and (n – 1) daughters during recovery. Live-cell liquid crystal polarized light microscope imaging showed nondisjunction was caused by chromosome malorientation. Whereas amphitely (sister kinetochore fibers to opposite poles) is normal, cold recovery induced anaphase syntely (sister fibers to the same pole) and merotely (fibers to both poles from 1 kinetochore). Maloriented chromosomes had stable metaphase positions near the equator or between the equator and a pole. Syntelics were at the spindle periphery at metaphase; their sisters disconnected at anaphase and moved all the way to a centrosome, as their strongly birefringent kinetochore fibers shortened. The kinetochore fibers of merotelics shortened little if any during anaphase, making anaphase lag common. If one fiber of a merotelic was more birefringent than the other, the less birefringent fiber lengthened with anaphase spindle elongation, often permitting inclusion of merotelics in a daughter nucleus. Meroamphitely (near amphitely but with some merotely) caused sisters to move in opposite directions. In contrast, syntely and merosyntely (near syntely but with some merotely) resulted in nondisjunction. Anaphase malorientations were more frequent after longer arrests, with particularly long arrests required to induce syntely and merosyntely.
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PreprintChromosomes can congress to the metaphase plate before biorientation( 2005-12-09) Kapoor, Tarun M. ; Lampson, Michael A. ; Hergert, Polla ; Cameron, Lisa ; Cimini, Daniela ; Salmon, Edward D. ; McEwen, Bruce F. ; Khodjakov, AlexeyThe stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle (i.e., “bi-oriented”). We found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy and correlative electron microscopy, we found that mono-oriented chromosomes could glide towards the spindle equator alongside kinetochore fibers attached to other already bi-oriented chromosomes. This congression mechanism depended on the kinetochore-associated plus enddirected microtubule motor CENP-E (kinesin-7).
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ArticleColloid osmotic parameterization and measurement of subcellular crowding(American Society for Cell Biology, 2019-01-14) Mitchison, Timothy J.Crowding of the subcellular environment by macromolecules is thought to promote protein aggregation and phase separation. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solutions that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solutions of macromolecules in contact with pores that are permeable to water and ions but not macromolecules. It generates depletion forces that push macromolecules together in crowded solutions and thus promotes aggregation and phase separation. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1–2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concentration–pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase separation biology, buffer design, and proteome evolution. I also discuss a pressure–tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.
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PreprintCompression regulates mitotic spindle length by a mechanochemical switch at the poles( 2009-05) Dumont, Sophie ; Mitchison, Timothy J.Although the molecules involved in mitosis are becoming better characterized, we still lack an understanding of the emergent mechanical properties of the mitotic spindle. For example, we cannot explain how spindle length is determined. To gain insight into how forces are generated and responded to in the spindle, we developed a method to apply controlled mechanical compression to metaphase mitotic spindles in living mammalian cells, while monitoring microtubules and kinetochores by fluorescence microscopy. Compression caused reversible spindle widening and lengthening to a new steadystate. Widening was a passive mechanical response, and lengthening an active mechanochemical process requiring microtubule polymerization but not kinesin-5 activity. Spindle morphology during lengthening and drug perturbations suggested that kinetochore fibers are pushed outwards by pole-directed forces generated within the spindle. Lengthening of kinetochore fibers occurred by inhibition of microtubule depolymerization at poles, with no change in sliding velocity, interkinetochore stretching, or kinetochore dynamics. We propose that spindle length is controlled by a mechanochemical switch at the poles that regulates the depolymerization rate of kinetochore-fibers in response to compression, and discuss models for how this switch is controlled. Poleward force appears to be exerted along kinetochore fibers by some mechanism other than kinesin-5 activity, and we speculate that it may arise from polymerization pressure from growing plus-ends of interpolar microtubules whose minus-ends are anchored in the fiber. These insights provide a framework for conceptualizing mechanical integration within the spindle.
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ArticleConditional knockout of the Menkes disease copper transporter demonstrates its critical role in embryogenesis(Public Library of Science, 2012-08-10) Wang, Yanfang ; Zhu, Sha ; Weisman, Gary A. ; Gitlin, Jonathan D. ; Petris, Michael J.The transition metal, copper (Cu), is an enzymatic cofactor required for a wide range of biochemical processes. Its essentiality is demonstrated by Menkes disease, an X-linked copper deficiency disorder characterized by defects in nervous-, cardiovascular- and skeletal systems, and is caused by mutations in the ATP7A copper transporter. Certain ATP7A mutations also cause X-linked Spinal Muscular Atrophy type 3 (SMAX3), which is characterized by neuromuscular defects absent an underlying systemic copper deficiency. While an understanding of these ATP7A-related disorders would clearly benefit from an animal model that permits tissue-specific deletion of the ATP7A gene, no such model currently exists. In this study, we generated a floxed mouse model allowing the conditional deletion of the Atp7a gene using Cre recombinase. Global deletion of Atp7a resulted in morphological and vascular defects in hemizygous male embryos and death in utero. Heterozygous deletion in females resulted in a 50% reduction in live births and a high postnatal lethality. These studies demonstrate the essential role of the Atp7a gene in mouse embryonic development and establish a powerful model for understanding the tissue-specific roles of ATP7A in copper metabolism and disease.
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ArticleDirectly probing the mechanical properties of the spindle and its matrix(Rockefeller University Press, 2010-02-22) Gatlin, Jesse C. ; Matov, Alexandre ; Danuser, Gaudenz ; Mitchison, Timothy J. ; Salmon, Edward D.Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.
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ArticleEna/VASP proteins have an anti-capping independent function in filopodia formation(American Society for Cell Biology, 2007-05-02) Applewhite, Derek A. ; Barzik, Melanie ; Kojima, Shin-ichiro ; Svitkina, Tatyana M. ; Gertler, Frank B. ; Borisy, Gary G.Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin–binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
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ArticleEntropy-driven formation of a chiral liquid-crystalline phase of helical filaments(American Physical Society, 2006-01-11) Barry, Edward ; Hensel, Zach ; Dogic, Zvonimir ; Shribak, Michael ; Oldenbourg, RudolfWe study the liquid-crystalline phase behavior of a concentrated suspension of helical flagella isolated from Salmonella typhimurium. Flagella are prepared with different polymorphic states, some of which have a pronounced helical character while others assume a rodlike shape. We show that the static phase behavior and dynamics of chiral helices are very different when compared to simpler achiral hard rods. With increasing concentration, helical flagella undergo an entropy-driven first order phase transition to a liquid-crystalline state having a novel chiral symmetry.
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ArticleA fiber-optic phase-randomizer for microscope illumination by laser(Rockefeller University Press, 1979-11) Ellis, Gordon W.
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ArticleFlexible and dynamic nucleosome fiber in living mammalian cells(Landes Bioscience, 2013-08-12) Nozaki, Tadasu ; Kaizu, Kazunari ; Pack, Chan-Gi ; Tamura, Sachiko ; Tani, Tomomi ; Hihara, Saera ; Nagai, Takeharu ; Takahashi, Koichi ; Maeshima, KazuhiroGenomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.
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PreprintForce and length in the mitotic spindle( 2009-07) Dumont, Sophie ; Mitchison, Timothy J.The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces. While molecular mechanisms that produce force have been described, our understanding of how they integrate with each other, and with the assembly-disassembly mechanisms that regulate length, is poor. We review current understanding of the basic architecture and dynamics of the metaphase spindle, and some of the elementary force producing mechanisms. We then discuss models for force integration, and spindle length determination. We also emphasize key missing data that notably includes absolute values of forces, and how they vary as a function of position, within the spindle.
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ArticleFunctional overlap of microtubule assembly factors in chromatin-promoted spindle assembly(American Society for Cell Biology, 2009-04-15) Groen, Aaron C. ; Maresca, Thomas J. ; Gatlin, Jesse C. ; Salmon, Edward D. ; Mitchison, Timothy J.Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, {gamma}-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either {gamma}-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.
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ArticleFunctional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy(American Society of Cell Biology, 2011-10-26) LaFountain, James R. ; Cohan, Christopher S. ; Oldenbourg, RudolfThe impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is “park,” in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.
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ArticleFungi 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.
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ArticleThe 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, SimonSeptins 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.