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Methods in Cell Biology

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https://www.readbyqxmd.com/read/28065322/visualization-of-cleavage-furrow-proteins-in-fixed-dividing-spermatocytes
#1
A Frappaolo, S Sechi, G Belloni, R Piergentili, M G Giansanti
Cytokinesis separates the cytoplasmic organelles and the duplicated genome into two daughter cells at the end of cell division. In animal cell cytokinesis, assembly and constriction of the contractile apparatus must be finely coordinated with plasma membrane remodeling and vesicle trafficking at the cleavage furrow. Accurate control of these events during cell cleavage is a fundamental task in all organisms and is also essential for maintaining ploidy and preventing neoplastic transformation. Drosophila male meiosis provides a well-suited cell system for exploring the molecular mechanisms underlying cytokinesis, combining the powerful tools of Drosophila genetics with unique cytological characteristics...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065321/studying-cytokinesis-in-drosophila-epithelial-tissues
#2
D Pinheiro, Y Bellaïche
Epithelial tissue cohesiveness is ensured through cell-cell junctions that maintain both adhesion and mechanical coupling between neighboring cells. During development, epithelial tissues undergo intensive cell proliferation. Cell division, and particularly cytokinesis, is coupled to the formation of new adhesive contacts, thereby preserving tissue integrity and propagating cell polarity. Remarkably, the geometry of the new interfaces is determined by the combined action of the dividing cell and its neighbors...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065320/imaging-cytokinesis-of-drosophila-s2-cells
#3
A Kechad, G R X Hickson
Animal cell cytokinesis proceeds through three successive stages: a contractile ring stage, an intercellular bridge stage, and an abscission stage. Many studies have identified a complex network of key proteins required for successful cytokinesis. While each component interacts with, and depends on, several other components, our understanding of how these proteins cooperate in space and time to ensure faithful progression through the stages of cytokinesis remains incomplete. A full understanding of the complexity of the process and its underlying machinery necessitates experimental systems that allow both genetic manipulation and real-time visualization of the various components throughout the successive stages of cytokinesis...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065319/xenopus-extract-approaches-to-studying-microtubule-organization-and-signaling-in-cytokinesis
#4
C M Field, J F Pelletier, T J Mitchison
We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ∼11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065318/in-vitro-reactivation-of-the-cytokinetic-contractile-ring-of-fission-yeast-cells
#5
I Mabuchi, J Kashiwazaki, M Mishra
Cytokinesis is a process by which a mother cell is divided into two daughter cells after chromosome segregation. In both animal and fungal cells, cytokinesis is carried out by the constriction of the contractile ring made up of actin, myosin-II, and other conserved proteins. Detailed genetic and cell biological analysis of cytokinesis has led to the identification of various genes involved in the process of cytokinesis including the cytological description of the process. However, detailed biochemical analysis of the process is lacking...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065317/preparation-of-centralspindlin-as-an-active-heterotetramer-of-kinesin-and-gtpase-activating-protein-subunits-for-in-vitro-structural-and-functional-assays
#6
M Mishima
Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the central spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the central spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission)...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065316/single-molecule-measurements-to-study-polymerization-dynamics-of-ftsz-ftsa-copolymers
#7
N Baranova, M Loose
Bacterial cytokinesis is commonly initiated by the Z-ring, a dynamic cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin-like GTPase, that like its eukaryotic relative forms protein filaments in the presence of GTP. Since the discovery of the Z-ring 25years ago, various models for the role of FtsZ have been suggested. However, important information about the architecture and dynamics of FtsZ filaments during cytokinesis is still missing. One reason for this lack of knowledge has been the small size of bacteria, which has made it difficult to resolve the orientation and dynamics of individual FtsZ filaments in the Z-ring...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065315/nuclear-displacement-and-fluorescence-recovery-after-photobleaching-frap-assays-to-study-division-site-placement-and-cytokinesis-in-fission-yeast
#8
P Ullal, P Bhatia, S G Martin
Cytokinesis is an essential cellular event that completes the cell division cycle. It begins with the assembly of an actomyosin contractile ring that undergoes constriction concomitant with the septum formation to divide the cell in two. Placement of the septum at the right position is important to ensure fidelity of the division process. In fission yeast, the medially placed nucleus is a major spatial cue to position the site of division. In this chapter, we describe a simple synthetic biology-based approach to displace the nucleus and study the consequence on division site positioning...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065314/an-active-contour-imagej-plugin-to-monitor-daughter-cell-size-in-3d-during-cytokinesis
#9
M B Smith, A Chaigne, E K Paluch
Controlling relative daughter cell size is key during cytokinesis. Uncontrolled size asymmetries can lead to aneuploidy and division failure. At the same time, precisely regulated size asymmetries are of crucial importance in many divisions during embryonic development. Therefore, being able to monitor daughter cell size is important in cytokinesis studies. However, freely available tools allowing to effectively measure the size of daughter cells in three dimensions during cytokinesis are missing. Here, we describe an open-access plugin for ImageJ or Fiji based on an active contour surface representation of the cells...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065313/cytokinesis-from-nanometers-to-micrometers-and-microseconds-to-minutes
#10
P Kothari, E S Schiffhauer, D N Robinson
Cytokinesis, a model cell shape change event, is controlled by an integrated system that coordinates the mitotic spindle signals with a mechanoresponsive cytoskeletal network that drives contractility and furrow ingression. Quantitative methods that measure cell mechanics, mechanoresponse (mechanical stress-induced protein accumulation), protein dynamics, and molecular interactions are necessary to provide insight into both the mechanical and biochemical components involved in cytokinesis and cell shape regulation...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065312/using-fast-acting-temperature-sensitive-mutants-to-study-cell-division-in-caenorhabditis-elegans
#11
T Davies, S Sundaramoorthy, S N Jordan, M Shirasu-Hiza, J Dumont, J C Canman
Fast-acting temperature-sensitive (ts) mutations are powerful conditional tools for studying transient cellular processes such as cytokinesis. Fast-acting ts cytokinesis-defective mutants are functional at the permissive temperature; yet show a fully penetrant loss-of-function cytokinesis failure phenotype when upshifted to the restrictive temperature. Fast-acting ts mutations thus allow functional tunability and rapid and reversible protein inactivation by simply shifting the temperature at precise times throughout cell division...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065311/variations-on-a-theme-imaging-cytokinetic-and-stable-rings-in-situ-using-caenorhabditis-elegans
#12
K Rehain, R A Green, K G Bourdages, A S Maddox
Cytokinesis is an essential event in canonical cell division. In multicellular organisms, cells must divide in the context of neighboring cells in intact tissues. Recent studies have shown that tissue architecture can regulate the dynamics of and molecular requirements for cytokinesis. On the other hand, regulated cytokinesis failure occurs in, and is required for the proper function of, certain cell types and tissues including cardiomyocytes, hepatocytes, and germ lines. One way to build our understanding of cytokinesis in diverse cell types is to visualize cytokinesis in intact tissues...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065310/analysis-of-postcytokinetic-roles-of-cytokinetic-components-in-caenorhabditis-elegans
#13
Y Chai, D Tian, W Li, G Ou
Cytokinesis requires the interplay between the cytoskeleton and plasma membrane. Emerging evidence indicates that some cytokinetic components are essential for the postcytokinetic events such as epithelium organization and neural development. We have recently developed live cell imaging and conditional knockout techniques to visualize cytokinetic proteins in Caenorhabditis elegans Q neuroblasts and separate their postcytokinetic functions from cytokinetic ones. Here we describe how the fluorescent reporter strains and conditional knockout C...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065309/analysis-of-protein-dynamics-during-cytokinesis-in-budding-yeast
#14
S Okada, C Wloka, E Bi
Cytokinesis is essential for development and survival of all organisms by increasing cell number and diversity. It is a highly regulated process that requires spatiotemporal coordination of hundreds of proteins functioning in the assembly, constriction, and disassembly of a contractile actomyosin ring, targeted vesicle fusion, and localized extracellular matrix remodeling. Cytokinesis has been studied in multiple systems with a wide range of technologies to learn the common principles. In this chapter, we describe the analysis of protein dynamics during cytokinesis in the budding yeast Saccharomyces cerevisiae by several live-cell imaging methods...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065308/studying-cytokinesis-and-midbody-remnants-using-correlative-light-scanning-em
#15
S Frémont, A Echard
Cytokinesis is an essential step of cell proliferation leading to the physical separation of the dividing cells. Cytokinesis relies on both large scale and local scale cell shape changes, and terminates with the final abscission cut that requires close apposition of the plasma membrane. While furrow ingression is a prominent feature of the early phase of cytokinesis and is easy to visualize in all models, from dividing eggs to culture cells, the later steps of cytokinesis until abscission can be much more difficult to visualize...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065307/analysis-of-cytokinesis-by-electron-microscopy
#16
J König, J Borrego-Pinto, D Streichert, M Munzig, P Lenart, T Müller-Reichert
Following up on a chapter on the Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis (MCB 79, 101-119), we present an adaptation of our established protocol for the ultrastructural analysis of either permeabilized or injected embryonic systems. We prepared both drug-treated early C. elegans embryos and fluorescently labeled sea urchin embryos of Lytechinus pictus for ultrastructural studies on animal cytokinesis. Here we focus on the initial preparation steps of postmitotic embryos for high-pressure freezing and subsequent electron microscopy with an emphasis on electron tomography...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065306/measuring-abscission-spatiotemporal-dynamics-using-quantitative-high-resolution-microscopy
#17
O Gershony, S Sherman, S Adar, I Segal, D Nachmias, I Goliand, N Elia
The spatiotemporal characteristics of ESCRT (Endosomal Sorting Complex Required for Transport)-mediated mammalian cytokinetic abscission have been studied in recent years using quantitative high-resolution light microscopy techniques. Here we describe how to apply spinning disk live cell imaging and structured illumination microscopy (SIM) to define the dynamics and structural organization of abscission and of proteins involved in abscission in a quantitative manner. We further provide a protocol to correlate the structural data, obtained by SIM, to the dynamic information obtained by live cell recordings...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065305/micromanipulation-of-daughter-cells-for-the-study-of-cytokinetic-abscission
#18
J Lafaurie-Janvore, C Lafaurie, M Piel
The last step of cytokinesis, abscission, consists in the severing of the intercellular bridge connecting the two daughter cells. Because daughter cells move randomly on regular cell culture substrates, the use of adhesive micropatterns facilitates the observation of the intercellular bridge and its severing. Here we propose general rules to design micropatterns optimized to study this process. In particular, these micropatterns allow a good stabilization of the daughter cells and a predictable positioning of the intercellular bridge...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065304/understanding-post-mitotic-roles-of-the-midbody-during-cell-differentiation-and-polarization
#19
E Peterman, R Prekeris
The midbody (MB) is a microtubule-rich structure that forms between dividing cells during final stages of cytokinesis. Previously thought to be a transient structure, MBs are now suggested to have additional roles beyond regulating cytokinesis. While the role MBs play during abscission are now well established, their function in regulating polarity and cell signaling are only beginning to be understood. Due to the newly found interest in the structure and functions of MBs, new techniques must be developed for further studies of this once-thought transient structure...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28065303/dynamics-of-sphingomyelin-and-cholesterol-enriched-lipid-domains-during-cytokinesis
#20
M Abe, T Kobayashi
Sphingomyelin (SM) and cholesterol (Chol) are the major lipids in the mammalian cells, which are mainly localized to the plasma membrane. Multiple lines of evidence suggest that these lipids form local lipid domains in the plasma membrane, playing functional roles in the cell. Several observations have suggested that these lipid domains are required for cytokinesis. In this chapter, we show the methods for visualizing SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins...
2017: Methods in Cell Biology
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