433 research outputs found
Targeting and transport : how microtubules control focal adhesion dynamics
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Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge
Analysis of focal adhesion turnover: a quantitative live-cell imaging example
No full textRecent advances in optical and fluorescent protein technology have rapidly raised expectations in cell biology, allowing quantitative insights into dynamic intracellular processes like never before. However, quantitative live-cell imaging comes with many challenges including how best to translate dynamic microscopy data into numerical outputs that can be used to make meaningful comparisons rather than relying on representative data sets. Here, we use analysis of focal adhesion turnover dynamics as a straightforward specific example on how to image, measure, and analyze intracellular protein dynamics, but we believe this outlines a thought process and can provide guidance on how to understand dynamic microcopy data of other intracellular structures
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INTERNALIZED DISTRIBUTED MOTOR COMMANDS BRIDGE ACTION AND COGNITION IN C. ELEGANS
No full textHere we find that the nematode C. elegans makes goal-oriented turns while foraging and can maintain a working memory of sensory activation prior to the execution of a turn. This information is integrated with body posture to localize appetitive stimuli. We construct a virtual-reality whole-brain imaging and neural perturbation system, and with it we find that this working memory is implemented by the coupled oscillation of two distributed neural motor command complexes. One complex decouples from motor output after sensory evidence accumulation and initiates turn execution; the second gates this process with a reliable time constant. We propose that the function of working memory via internalization of motor oscillations could represent the evolutionary origin of internal neural processing i.e. thought, and a foundation of higher cognition
Imaging intracellular protein dynamics by spinning disk confocal microscopy
No full textThe palette of fluorescent proteins (FPs) has grown exponentially over the past decade, and as a result, live imaging of cells expressing fluorescently tagged proteins is becoming more and more mainstream. Spinning disk confocal (SDC) microscopy is a high-speed optical sectioning technique and a method of choice to observe and analyze intracellular FP dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low-noise scientific grade-cooled charge-coupled device cameras, and can achieve frame rates of up to 1000 frames per second. In this chapter, we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy and provide a rationale for specific design choices. We also give guidelines of how other imaging techniques such as total internal reflection microscopy or spatially controlled photoactivation can be coupled with SDC imaging and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction
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Role of CLASP2 phosphorylation in regulating kinetochore-microtubule interactions
No full textProper chromosome segregation requires dynamic regulation of kinetochore-microtubule attachments throughout mitosis. Multiple kinetochore proteins display microtubule-binding activity, yet how exactly these proteins are spatially and temporally regulated is unclear. Cytoplasmic linker-associating proteins (CLASPs) are present in the outer kinetochore, in close proximity to microtubule ends, and are required for mitosis. Here, we test whether phosphorylation of CLASP2 during mitosis serves as a mechanism to regulate kinetochore-microtubule interactions and the fidelity of chromosome segregation. We show that cyclin-dependent kinase 1 (Cdk1) and glycogen synthase kinase beta; (GSK3beta;)-dependent mitotic phosphorylation of CLASP2 within its microtubule end-binding domain inhibits its microtubule end-binding activity but does not affect CLASP2 kinetochore binding. Deregulation of CLASP2 phosphorylation does not affect initial chromosome congression, but weakens kinetochore-microtubule attachments. In the presence of endogenous CLASP2, phosphorylation-deficient CLASP2 increases average interkinetochore distance, while phosphomimetic CLASP2 decreases average interkinetochore distance measurements. Together, these results suggest that CLASP2 microtubule binding at the kinetochore is required for stable, tension-generating kinetochore-microtubule attachments. Furthermore, cells expressing CLASP2 phosphomutants have abnormal kinetochore dynamics and in some cells, kinetochore pairs flip about the metaphase plate, suggesting absent or imbalanced microtubule attachments. Expression of either nonphosphorylatable or phosphomimetic CLASP2 fails to rescue depletion of CLASP2 and ultimately leads to an increase in lagging chromosomes. Together, these results characterize specific phosphorylation sites in CLASP2 that negatively regulate microtubule binding and present a novel mechanism of Cdk1 and GSK3beta;-mediated control of kinetochore-microtubule interactions
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Function and dynamics of microtubules and plus-end-tracking proteins during 3D epithelial remodeling
No full textEpithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays essential roles in development and disease of multicellular organisms. How cytoskeleton dynamics, especially microtubule dynamics, are controlled and contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We optimize confocal live-cell imaging to analyze microtubule function and dynamics during 3D epithelial remodeling of polarized Madin-Darby kidney epithelial cells that undergo a partial epithelial-to-mesenchymal transition (pEMT) in response to hepatocyte growth factor (HGF). We found that cellular extensions at the basal surface of HGF-treated cysts are densely packed with microtubules. Computational tracking of EB1-2xEGFP showed large numbers of microtubules growing persistently from the apical domain into these extensions and an increase in microtubule growth rate in response to HGF. We tested the role of microtubule plus-end-tracking protein (+TIP) complexes in 3D epithelial remodeling by depleting cells of EB1, an adaptor protein that mediates recruitment of other +TIP proteins to growing microtubule plus ends. In EB1-depleted cells, microtubules displayed rapid lateral and retrograde movements demonstrating that EB1 is required to stabilize and organize microtubules in HGF-induced extensions. EB1-depleted cysts formed shorter, more branched, extensions suggesting that EB1 is required for productive HGF-induced extension outgrowth. Analysis of cell-matrix interactions and F-actin dynamics revealed that control extensions progressively pulled on and deformed the extracellular matrix (ECM) typically with one F-actin-rich protrusion near the cell tip with cell-matrix adhesions that turned over in a coordinated fashion. In contrast, EB1-depleted cells produced multiple highly dynamic protrusions with nascent adhesions that were uncoordinated, mislocalized and did not productively engage the matrix. As a result EB1-depleted extensions rapidly protruded, retracted and changed direction. Finally we show that trafficking of VAMP3-positive vesicles to the protrusion tip is disrupted in EB1-depleted cells. Together these data suggest that EB1-mediated organization of the MT cytoskeleton and associated vesicle delivery to the tip of HGF-induced extensions are likely required to coordinate cell-matrix adhesion and protrusion dynamics during 3D epithelial remodeling. It will be important in the future to investigate a broader role for EB1-mediated +TIP complexes in other normal and disease states of epithelial remodeling
Map Involved in Spindle Pole Organization
No full textTPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles
EBs clip CLIPs to growing microtubule ends
No full textProteins that track growing microtubule (MT) ends are important for many aspects of intracellular MT function, but the mechanism by which these +TIPs accumulate at MT ends has been the subject of a long-standing controversy. In this issue, Bieling et al. (Bieling, P., S. Kandels-Lewis, I.A. Telley, J. van Dijk, C. Janke, and T. Surrey. 2008. J. Cell Biol. 183:1223-1233) reconstitute plus end tracking of EB1 and CLIP-170 in vitro, which demonstrates that CLIP-170 plus end tracking is EB1-dependent and that both +TIPs rapidly exchange between a soluble and a plus end-associated pool. This strongly supports the hypothesis that plus end tracking depends on a biochemical property of growing MT ends, and that the characteristic +TIP comets result from the generation of new +TIP binding sites through MT polymerization in combination with the exponential decay of these binding sites
Beck, Rose Marie & Frank Wittmann (eds.) 2004. African Media Cultures – Transdisciplinary Perspectives. Cultures de médias en Afrique. Perspectives transdisciplinaires - Topics in African Studies, Vol. 2, Köln: Köppe Verlag, 320 pages, 2 b/w photos, 17 tables, 15 graphs, €34,80
No full textBeck and Wittmann\u27s volume is a most welcome contribution about types and uses of traditional and modern media in Africa. Their volume is divided into fifteen chapters preceded by a comprehensive introduction and followed by some information about the author
Boreomysis bispinosa O. S. Tattersall 1955
No full text<i>Boreomysis bispinosa</i> O.S. Tattersall, 1955 <p> <b>Material examined</b> (non-types only) ANGOLA BASIN • 1 imm. (BL = 8.3 mm, eyes missing); 17°4.935′ S, 4°40.805′ E to 17°07.454′ S,</p> <p>4°42.276′ E; bottom depth 5460– 5460 m; 25 Jul. 2000; DIVA-1 exped., #344; epinet of epibenthic sledge; ZMH 58248 • 1 ♀ ad. (estimated BL = 12.6 mm, cephalothorax and exuvia); 16°16.989′ S, 5°27.279′ E to 16°19.280′ S, 5°27.205′ E; bottom depth 5430–5433 m; 28 Jul. 2000; DIVA-1 exped., #348; supranet of epibenthic sledge; ZMH 58249.</p> Type locality <p>Not stated by O.S. Tattersall (1955). On page 14 she indicated a “female type ” taken off Cape Town, depth 1350 – 1250 m, and a “male type ” NE of St. Helena, depth 1450 – 700 m. A rough estimate by the present author suggests that the stations are from 34° S, 17° E and 15° S, 5° W, respectively.</p> Distribution <p> Previously reported from the Atlantic Ocean and from the Atlantic sector of the Southern Ocean, 51° N– 54° S, 36° W– 17° E (O.S. Tattersall 1955; Mauchline & Murano 1977; Hargreaves 1997; Wittmann <i>et al.</i> 2004; Petryashov 2005 b; San Vicente 2011). The animals were sampled with benthic as well as pelagic gears. The here documented records in the SE-Atlantic at 16– 17° S, 5° E are within the already known geographical range, while the bathymetrical range of 700–4050 m is now extended down to 5430–5460 m (see also Discussion).</p>Published as part of <i>Wittmann, Karl J., 2020, Lophogastrida and Mysida (Crustacea) of the " DIVA- 1 " deep-sea expedition to the Angola Basin (SE-Atlantic), pp. 1-43 in European Journal of Taxonomy 628</i> on page 15, DOI: 10.5852/ejt.2020.628, <a href="http://zenodo.org/record/3756146">http://zenodo.org/record/3756146</a>
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