scholarly journals Microtubules control cellular shape and coherence in amoeboid migrating cells

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Aglaja Kopf ◽  
Jörg Renkawitz ◽  
Robert Hauschild ◽  
Irute Girkontaite ◽  
Kerry Tedford ◽  
...  
Keyword(s):  
Cell Shape ◽  
Shape Control ◽  
Microtubule Depolymerization ◽  
Microtubule Organizing Center ◽  
Amoeboid Cell ◽  
Actomyosin Contractility ◽  
Organizing Center ◽  
A Cell ◽  
And Control ◽  
Rate Limit

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.

scholarly journals Microtubules control cellular shape and coherence in amoeboid migrating cells

2019 ◽  
Author(s):  
Aglaja Kopf ◽  
Jörg Renkawitz ◽  
Robert Hauschild ◽  
Irute Girkontaite ◽  
Kerry Tedford ◽  
...  
Keyword(s):  
Cell Shape ◽  
Shape Control ◽  
Complex Geometry ◽  
Control Cell ◽  
Three Dimensional ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell ◽  
Cell Shape Control ◽  
Rate Limit

Cells navigating through tissues face a fundamental challenge: while multiple cellular protrusions explore different paths through the complex geometry of an interstitial matrix the cell needs to avoid becoming too long or ramified, which might ultimately lead to a loss of physical coherence. How a cell surveys its own shape to inform the actomyosin system to retract entangled or stretched protrusions is not understood. Here, we demonstrate that spatially distinct microtubule (MT) dynamics regulate amoeboid cell migration by locally specifying the retraction of explorative protrusions. In migrating dendritic cells (DCs), the microtubule organizing center (MTOC) guides the path through a three dimensional (3D) interstitium and local MT depolymerization in protrusions remote from the MTOC triggers myosin II dependent contractility via the RhoA exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: i) impaired cell edge coordination during path-finding and ii) defective adhesion-resolution. Such compromised cell shape control is particularly hindering when cells navigate through geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. Our data demonstrate that MTs control cell shape and coherence by locally controlling protrusion-retraction dynamics of the actomyosin system.

scholarly journals Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

2012 ◽  
Vol 12 (2) ◽  
pp. 265-277 ◽  
Author(s):  
Julia D. Romano ◽  
Catherine de Beaumont ◽  
Jose A. Carrasco ◽  
Karen Ehrenman ◽  
Patrik M. Bavoil ◽  
...  
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Productive Infection ◽  
Microtubule Organizing Center ◽  
Nutrient Pools ◽  
Content Type ◽  
Cell Structures ◽  
Organizing Center ◽  
A Cell ◽  
Fierce Competition

ABSTRACTThe prokaryoteChlamydia trachomatisand the protozoanToxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similarmodus operandito colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected byC. trachomatisandT. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and thatToxoplasmaholds a significant competitive advantage overChlamydia. Here we have expanded our coinfection studies by examining the respective abilities ofChlamydiaandToxoplasmato co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However,ToxoplasmaoutcompetesChlamydiato the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected withChlamydia, the centrosomes become associated with the chlamydial inclusion, while theToxoplasmaparasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by bothChlamydiaandToxoplasmadepends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells

1997 ◽  
Vol 110 (19) ◽  
pp. 2391-2401 ◽  
Author(s):  
A.M. Yvon ◽  
P. Wadsworth
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Human Kidney ◽  
Light Level ◽  
Pause Duration ◽  
Living Cells ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

scholarly journals STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles

2018 ◽  
Vol 115 (10) ◽  
pp. E2246-E2253 ◽  
Author(s):  
Rifka Vlijm ◽  
Xue Li ◽  
Marko Panic ◽  
Diana Rüthnick ◽  
Shoji Hata ◽  
...  
Keyword(s):  
Cell Migration ◽  
Mitotic Spindle ◽  
Stimulated Emission ◽  
Microtubule Organizing Center ◽  
Spindle Formation ◽  
Spectrin Repeat ◽  
Cilia Formation ◽  
Organizing Center ◽  
A Cell ◽  
Vast Network

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.

Microtubule depolymerization inhibits transport of cathepsin D from the Golgi apparatus to lysosomes

1990 ◽  
Vol 96 (4) ◽  
pp. 711-720
Author(s):  
J. Scheel ◽  
R. Matteoni ◽  
T. Ludwig ◽  
B. Hoflack ◽  
T.E. Kreis
Keyword(s):  
Golgi Apparatus ◽  
Cathepsin D ◽  
Lysosomal Enzymes ◽  
Spatial Organization ◽  
Microtubule Depolymerization ◽  
Microtubule Organizing Center ◽  
Microtubule Network ◽  
Organizing Center ◽  
Efficient Transport ◽  
Golgi Network

Lysosomes as well as a prelysosomal compartment rich in the mannose 6-phosphate receptor are clustered close to the Golgi apparatus in the perinuclear region of the microtubule organizing center in interphase human skin fibroblasts. The spatial organization of these organelles depends on an intact microtubule network. Depolymerization of the microtubules by treatment of cells with nocodazole leads to random scattering of Golgi elements, the prelysosomal compartment, and lysosomes throughout the cytoplasm. To test whether microtubules and the spatial organization of these organelles are important for efficient transport of lysosomal enzymes, the effect of microtubule depolymerization on the maturation of newly synthesized cathepsin D was studied. An up to fivefold inhibition of proteolytic maturation of cathepsin D was observed in drug-treated cells. This effect was due to a decreased rate of transport of cathepsin D from the Golgi apparatus to lysosomes. Depolymerization of microtubules did not inhibit transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi network. Furthermore, synthesis of the phosphomannosyl marker present on cathepsin D was not affected by nocodazole. These results suggest that efficient transport of cathepsin D from the Golgi apparatus to a prelysosomal compartment and lysosomes is facilitated by microtubules and the spatial organization of these organelles.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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