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Methods in Cell Biology

N F Valadares, R C Garratt
Septins are filament-forming proteins found in many eukaryotes. Despite being important components of the cytoskeleton, only recently details of their macromolecular assemblies and crystal structures have started to appear in the literature. These are of fundamental importance to the understanding of cytoskeleton dynamics, membrane barrier formation, and bacterial caging, as well as essential cellular processes such as cell division, exocytosis, and vesicle trafficking. However, obtaining this data is frequently hindered by several experimental difficulties common to the majority of septin samples...
2016: Methods in Cell Biology
L R Heasley, M A McMurray
Progress on the study of the molecular and cellular biology of septins would be greatly accelerated by the development of small molecules that directly inhibit higher-order septin assembly in vivo. By comparison, molecules like latrunculin, paclitaxil, benomyl, etc. allow researchers to acutely perturb the actin or tubulin cytoskeletal networks. Two small molecules, forchlorfenuron (FCF; N-(2-chloro-4pyridyl)-N-phenylurea) and 1-ethyl-3-(4-methoxyphenyl)-6-methylpyrimido[5,4-e][1,2,4]triazine-5,7-dione (PubChem CID 906558), have documented effects on septin localization and/or function, although for each molecule there is also strong evidence for off-target effects...
2016: Methods in Cell Biology
H Ito, R Morishita, H Tabata, K Nagata
Correct neuronal migration is crucial for the brain architecture and function. During brain development, excitatory and inhibitory neurons generated in the ventricular zone (VZ) of the dorsal telencephalon and ganglionic medial eminence, respectively, move to their final destinations in tightly regulated spatiotemporal manners. While a variety of morphological methods have been applied to neurobiology, in utero electroporation (IUE) technique is one of the most powerful tools for rapid gain- and loss-of-function studies of brain development...
2016: Methods in Cell Biology
L K Parajuli, N Ageta-Ishihara, H Ageta, Y Fukazawa, M Kinoshita
The minimal functional units of the mammalian septin system are diverse heterooligomers of SEPT1-14 subunits, which are most abundantly and differentially expressed in postmitotic neurons and glia. The subunit compositions of such heterooligomers are thought to differentiate their affinity for other proteins and lipids, and subcellular localization. Thus, high-precision quantification and mapping of each subunit is necessary to understand their subcellular functions and physiological roles. However, systematic information on the localization of individual septin subunits in the mammalian nervous system is limited...
2016: Methods in Cell Biology
M S Kim, C D Froese, H Xie, W S Trimble
Primary cilia are cellular antennae that receive and transduce extracellular cues. These microtubule-rich structures are comprised of at least three distinct ciliary compartments: basal bodies, transition zone, and axoneme. Septins have been implicated in cilia function at the transition zone, but accumulating evidence suggests that they localize predominantly within the axoneme. Here, we describe three fixation conditions that preserve the substructure of primary cilia and demonstrate known ciliary proteins that localize to these distinct ciliary substructures...
2016: Methods in Cell Biology
E T Spiliotis, E P Karasmanis, L Dolat
Septins are a major component of the mammalian cytoskeleton. Septins associate with filamentous actin (F-actin) and microtubules, but the nature and significance of these interactions are not well understood. Fluorescence microscopy of F-actin- and microtubule-associated septins in fixed and living cells has been instrumental in uncovering septin functions in cellular morphogenesis and cytoskeleton-dependent processes (eg, cell division, cell migration). Here, we provide a detailed methodology for the visualization of endogenous septins by immunofluorescence microscopy, discussing sample preparation and reagents that are critical for optimal staining...
2016: Methods in Cell Biology
A Willis, M Mazon-Moya, S Mostowy
The zebrafish (Danio rerio) is an important animal model to study cell biology in vivo. Benefits of the zebrafish include a fully annotated reference genome, an easily manipulable genome (for example, by morpholino oligonucleotide or CRISPR-Cas9), and transparent embryos for noninvasive, real-time microscopy using fluorescent transgenic lines. Zebrafish have orthologues of most human septins, and studies using larvae were used to investigate the role of septins in vertebrate development. The zebrafish larva is also an established model to study the cell biology of infection and has recently been used to visualize septin assembly during bacterial infection in vivo...
2016: Methods in Cell Biology
M Mavrakis, F-C Tsai, G H Koenderink
Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy...
2016: Methods in Cell Biology
M Mavrakis
Functional studies in Drosophila have been key for establishing a role for the septin family of proteins in animal cell division and thus extending for the first time observations from the budding yeast to animal cells. Visualizing the distribution of specific septins in different Drosophila tissues and, in particular, in the Drosophila embryo, together with biochemical and mutant phenotype data, has contributed important advances to our understanding of animal septin biology, suggesting roles in processes other than in cytokinesis...
2016: Methods in Cell Biology
C Kaplan, C Yu, H Ewers
Heteromeric complexes of GTP-binding proteins from the septin family assemble into higher order structures that are essential for cell division in many organisms. The correct organization of the subunits into filaments, gauzes, and rings is the basis of septin function in this process. Electron microscopy and polarization fluorescence microscopy contributed greatly to the understanding of the dynamics and organization of such structures. However, both methods show technical limitations in resolution and specificity that do not allow the identification of individual septin complexes in assemblies in intact cells...
2016: Methods in Cell Biology
S Baumann, S Zander, S Weidtkamp-Peters, M Feldbrügge
Septins are highly conserved cytoskeletal proteins involved in a variety of biological processes such as cell polarization and cytokinesis. In humans, functional defects in these proteins have been linked to cancer and neuronal diseases. In recent years, substantial progress has been made in studying the structure of septin subunits and the formation of defined heteromeric building blocks. These are assembled into higher-order structures at distinct subcellular sites. An important microscopic approach in studying septin assembly and dynamics is the use of septins tagged with fluorescent proteins...
2016: Methods in Cell Biology
A Smith, M Momany
Individual septins from separate phylogenetic groups assemble into heteropolymer rods with defined compositions. Heteropolymer rods can in turn assemble into larger higher-order structures (HOS). Using a combination of immunoprecipitation and fluorescence microscopy in Aspergillus nidulans wild-type and septin-mutant strains, we have shown that even when septin HOS are not visible by fluorescence microscopy, heteropolymer rods can still be present and that a septin found largely in filamentous fungi is required for a subset of HOS...
2016: Methods in Cell Biology
S Krokowski, S Mostowy
Investigation of the host cytoskeleton during infection by bacterial pathogens has significantly contributed to our understanding of cell biology and host defense. Work has shown that septins are recruited to the phagocytic cup as collarlike structures and enable bacterial entry into host cells. In the cytosol, septins can entrap actin-polymerizing bacteria in cage-like structures for targeting to autophagy, a highly conserved intracellular degradation process. In this chapter, we describe methods to investigate septin assembly and function during infection by bacterial pathogens...
2016: Methods in Cell Biology
M A McMurray
In Saccharomyces cerevisiae, septin mutations have severe effects on colony-forming ability, particularly at high temperatures, allowing the full variety of genetic tools available in this model organism to be applied to the study of septin biology. Although many details of septin function remain unknown, one can exploit a small number of easily scored phenotypes-proliferation capacity, cell morphology, septin localization, and septin ring integrity-as sensitive readouts of properly assembled septin filaments...
2016: Methods in Cell Biology
K Ong, T Svitkina, E Bi
Septins are cytoskeletal proteins involved in diverse biological processes including cytokinesis, cell morphogenesis, motility, and ciliogenesis. Septins form various filamentous structures in vitro and in vivo, but the higher-order architecture of septin structures in vivo remains poorly defined. The best understood system in this respect is the budding yeast Saccharomyces cerevisiae, where septins form a ring structure that undergoes multiple stages of remodeling during the cell cycle. In this chapter, we describe a method for visualizing supramolecular septin structures in yeast at high spatial resolution using platinum replica electron microscopy...
2016: Methods in Cell Biology
A A Bridges, A S Gladfelter
Septins are polymerizing eukaryotic proteins that play conserved roles in cell cortex organization and are essential in many cell types. How septin dynamics and protein-protein interactions determine their function at the plasma membrane remains a mystery. Here, we present a method for recapitulating septin polymerization and lipid interaction utilizing supported lipid bilayers to mimic the eukaryotic plasma membrane. Septins on supported lipid bilayers can be visualized with single-molecule sensitivity using total internal reflective fluorescence microscopy...
2016: Methods in Cell Biology
E A Booth, J Thorner
Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions...
2016: Methods in Cell Biology
A Bertin, E Nogales
Septins are highly conserved and essential eukaryotic cytoskeletal proteins that interact with the inner plasma membrane. They are involved in essential functions requiring cell membrane remodeling and compartmentalization, such as cell division and dendrite morphogenesis, and have been implicated in numerous diseases. Depending on the organisms and on the type of tissue, a specific set of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into linear, symmetric rods that can further organize into linear filaments several microns in length...
2016: Methods in Cell Biology
M Onishi, J R Pringle
We have confirmed and extended previous reports of a wide distribution of septin proteins in the eukaryotic phylogeny. It now appears that septins are present in at least some representatives of every eukaryotic supergroup, with the possible exception of the Excavata. Presently, almost nothing is known of the structure, assembly, and biological roles of septins outside of the opisthokonts (animals, fungi, and their close relatives). Thus, studies of the septins in the highly diverse and distantly related nonopisthokont groups present a major opportunity to gain a much deeper understanding of septin core function and evolution, and we discuss briefly the excellent prospects for capitalizing on this opportunity in the next few years...
2016: Methods in Cell Biology
Z M Varga, K N Murray
In this chapter we review the components of the fish health program at the Zebrafish International Resource Center. We describe health-monitoring strategies to assess individual and colony health, practices to prevent the spread of pathogens within the fish colony, and a biosecurity program designed to prevent entry of new fish pathogens. While this program is designed for a facility on a recirculating water system with expectations of high volumes of import and export, many of the components can be directly applied or modified for application in facilities of different sizes and with other programmatic goals...
2016: Methods in Cell Biology
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