scholarly journals Translocation and clustering of endosomes and lysosomes depends on microtubules.

1987 ◽  
Vol 105 (3) ◽  
pp. 1253-1265 ◽  
Author(s):  
R Matteoni ◽  
T E Kreis
Keyword(s):  
Rat Kidney ◽  
Living Cells ◽  
Cytochalasin D ◽  
Microtubule Organizing Center ◽  
Enhanced Fluorescence ◽  
Organelle Movement ◽  
Organizing Center ◽  
Normal Rat ◽  
Immunofluorescence Labeling

Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recognizing a lysosomal glycoprotein (LGP 120; Lewis, V., S.A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) reveals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of lysosomes depends on microtubules. When the interphase microtubules are depolymerized by treatment of the cells with nocodazole or during mitosis, the lysosomes disperse throughout the cytoplasm. Lysosomes recluster rapidly (within 30-60 min) in the region of the centrosomes either upon removal of the drug, or, in telophase, when repolymerization of interphase microtubules has occurred. During this translocation process the lysosomes can be found aligned along centrosomal microtubules. Endosomes and lysosomes can be visualized by incubating living cells with acridine orange. We have analyzed the movement of these labeled endocytic organelles in vivo by video-enhanced fluorescence microscopy. Translocation of endosomes and lysosomes occurs along linear tracks (up to 10 microns long) by discontinuous saltations (with velocities of up to 2.5 microns/s). Organelles move bidirectionally with respect to the MTOC. This movement ceases when microtubules are depolymerized by treatment of the cells with nocodazole. After nocodazole washout and microtubule repolymerization, the translocation and reclustering of fluorescent organelles predominantly occurs in a unidirectional manner towards the area of the MTOC. Organelle movement remains unaffected when cells are treated with cytochalasin D, or when the collapse of intermediate filaments is induced by microinjected monoclonal antivimentin antibodies. It can be concluded that translocation of endosomes and lysosomes occurs along microtubules and is independent of the intermediate filament and microfilament networks.

scholarly journals Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae ofAshbya gossypii

2010 ◽  
Vol 21 (1) ◽  
pp. 18-28 ◽  
Author(s):  
Claudia Lang ◽  
Sandrine Grava ◽  
Tineke van den Hoorn ◽  
Rhonda Trimble ◽  
Peter Philippsen ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Hyphal Growth ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Organizing Center ◽  
Cytoplasmic Side ◽  
Independent Mode

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

scholarly journals Regulation of myonuclear positioning and muscle function by the skeletal muscle-specific CIP protein

2020 ◽  
Vol 117 (32) ◽  
pp. 19254-19265
Author(s):  
Jianming Liu ◽  
Zhan-Peng Huang ◽  
Mao Nie ◽  
Gang Wang ◽  
William J. Silva ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Function ◽  
Neuromuscular Junctions ◽  
Linc Complex ◽  
Muscle Dystrophy ◽  
Microtubule Organizing Center ◽  
Anchoring Protein ◽  
Organizing Center ◽  
Genetic Deletion

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy inmdxmice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.

Imaging of plasmacytoid dendritic cell interactions with T cells

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 75-84 ◽  
Author(s):  
María Mittelbrunn ◽  
Gloria Martínez del Hoyo ◽  
María López-Bravo ◽  
Noa B. Martín-Cofreces ◽  
Alix Scholer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Plasmacytoid Dendritic Cell ◽  
Time Lapse ◽  
Cell Interactions ◽  
Type I ◽  
Microtubule Organizing Center ◽  
Immune Synapse ◽  
Organizing Center

Abstract Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4+ T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-θ, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC–T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

scholarly journals 3′-Untranslated regions of oxidative phosphorylation mRNAs function in vivo as enhancers of translation

2000 ◽  
Vol 352 (1) ◽  
pp. 109-115 ◽  
Author(s):  
Carlo M. Di LIEGRO ◽  
Marianna BELLAFIORE ◽  
José M. IZQUIERDO ◽  
Anja RANTANEN ◽  
José M. CUEZVA
Keyword(s):  
Oxidative Phosphorylation ◽  
Atp Synthase ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
In Vitro Translation ◽  
Mitochondrial Transcription Factor ◽  
Normal Rat ◽  
Green Fluorescent

Recent findings have indicated that the 3´-untranslated region (3´-UTR) of the mRNA encoding the β-catalytic subunit of the mitochondrial H+-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255–5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849–855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3´-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3´-UTR of the mRNAs encoding the α- and β-subunits of the rat H+-ATP synthase complex, as well as in subunit IV of cytochrome c oxidase. No TEA is present in the 3´-UTR of the somatic mRNA encoding rat mitochondrial transcription factor A. Interestingly, the TEA of the 3´-UTR of mRNAs of oxidative phosphorylation is different, depending upon the cell type analysed. These data provide the first in vivo evidence of a novel cell-specific mechanism for the control of the translation of mRNAs required in mitochondrial function.

scholarly journals Single-Particle Tracking Porcine Epidemic Diarrhea Virus Moving along Microtubules in Living Cells

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 124
Author(s):  
Yangyang Li ◽  
Wei Hou ◽  
Jian Wang ◽  
Fei Liu
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Porcine Epidemic Diarrhea Virus ◽  
Living Cells ◽  
Single Particle Tracking ◽  
Microtubule Organizing Center ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Organizing Center ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused severe damage to the swine industry. Although viruses are believed to hijack the microtubule-based transport system, the exact manner of PEDV moving along microtubules has not been fully characterized. In this study, PEDV was labeled with quantum dots which have great brightness and photostability. By using quantum dot-labeled PEDV and single-particle tracking, we were able to systematically dissect the dynamic behaviors of PEDV moving along the microtubules in living cells. We found that PEDVs maintained a restricted motion mode with a relatively stable speed in the cell membrane region while displaying a slow–fast–slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule-organizing center. The return movements of small amounts of PEDVs were also observed in living cells. Collectively, our work is crucial for understanding the movement of PEDV in living cells; the proposed work also provides important references for further analysis and studies of the infection mechanism of PEDV.

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 Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells.

1995 ◽  
Vol 131 (6) ◽  
pp. 1649-1658 ◽  
Author(s):  
M R Jacobson ◽  
L G Cao ◽  
Y L Wang ◽  
T Pederson
Keyword(s):  
Rat Kidney ◽  
Living Cells ◽  
Dense Fibrillar Component ◽  
Optical Sectioning ◽  
Sequence Element ◽  
Dynamic Localization ◽  
Rnase Mrp ◽  
Stereo Reconstruction ◽  
Normal Rat ◽  
Fibrillar Component

The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These results demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA.

scholarly journals Visualization of the intracellular behavior of HIV in living cells

2002 ◽  
Vol 159 (3) ◽  
pp. 441-452 ◽  
Author(s):  
David McDonald ◽  
Marie A. Vodicka ◽  
Ginger Lucero ◽  
Tatyana M. Svitkina ◽  
Gary G. Borisy ◽  
...  
Keyword(s):  
Life Cycle ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Network ◽  
Organizing Center ◽  
Infected Cells ◽  
Resolution Imaging ◽  
Transcription Complexes ◽  
Hiv 1

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

scholarly journals Cytoplasmic motions, rheology, and structure probed by a novel magnetic particle method.

1985 ◽  
Vol 101 (1) ◽  
pp. 130-140 ◽  
Author(s):  
P A Valberg ◽  
D F Albertini
Keyword(s):  
Magnetic Particles ◽  
Particle Method ◽  
Time Lapse ◽  
Living Cells ◽  
Cellular Motility ◽  
Phagocytic Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
Pulmonary Macrophages ◽  
Field Decay

The motions of magnetic particles contained within organelles of living cells were followed by measuring magnetic fields generated by the particles. The alignment of particles was sensed magnetometrically and was manipulated by external fields, allowing non-invasive detection of particle motion as well as examination of cytoplasmic viscoelasticity. Motility and rheology data are presented for pulmonary macrophages isolated from lungs of hamsters 1 d after the animals had breathed airborne gamma-Fe2O3 particles. The magnetic directions of particles within phagosomes and secondary lysosomes were aligned, and the weak magnetic field produced by the particles was recorded. For dead cells, this remanent field was constant, but for viable macrophages, the remanent field decreased rapidly so that only 42% of its initial magnitude remained 5 min after alignment. A twisting field was applied perpendicular to the direction of alignment and the rate at which particles reoriented to this new direction was followed. The same twisting was repeated for particles suspended in a series of viscosity standards. Based on this approach, the low-shear apparent intracellular viscosity was estimated to be 1.2-2.7 X 10(3) Pa.s (1.2-2.7 X 10(4) poise). Time-lapse video microscopy confirmed the alignment of ingested particles upon magnetization and showed persistent cellular motility during randomization of alignment. Cytochalasin D and low temperature both reduced cytoplasmic activity and remanent-field decay, but affected rheology differently. Magnetic particles were observed in association with the microtubule organizing center by immunofluorescence microscopy; magnetization did not affect microtubule distribution. However, both vimentin intermediate filaments and f-actin reorganized after magnetization. These data demonstrate that magnetometry of isolated phagocytic cells can probe organelle movements, rheology, and physical properties of the cytoskeleton in living cells.

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