Microtubule Dynamics and Organelle Transport in Reticulomyxa

1990 ◽  
Vol 48 (3) ◽  
pp. 194-195
Author(s):  
Yih-Tai Chen ◽  
Ursula Euteneuer ◽  
Ken B. Johnson ◽  
Michael P. Koonce ◽  
Manfred Schliwa
Keyword(s):  
Plasma Membrane ◽  
Light Microscopy ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Model Systems ◽  
Organelle Transport ◽  
Biochemical Characteristics ◽  
Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

scholarly journals Dynein efficiently navigates the dendritic cytoskeleton to drive the retrograde trafficking of BDNF/TrkB signaling endosomes

2017 ◽  
Vol 28 (19) ◽  
pp. 2543-2554 ◽  
Author(s):  
Swathi Ayloo ◽  
Pedro Guedes-Dias ◽  
Amy E. Ghiretti ◽  
Erika L. F. Holzbaur
Keyword(s):  
Real Time ◽  
Axonal Transport ◽  
Hippocampal Neurons ◽  
Cytoplasmic Dynein ◽  
Microtubule Dynamics ◽  
Neuronal Function ◽  
Retrograde Trafficking ◽  
Basic Understanding ◽  
Dependent Transport ◽  
Dynamic Microtubule

The efficient transport of cargoes within axons and dendrites is critical for neuronal function. Although we have a basic understanding of axonal transport, much less is known about transport in dendrites. We used an optogenetic approach to recruit motor proteins to cargo in real time within axons or dendrites in hippocampal neurons. Kinesin-1, a robust axonal motor, moves cargo less efficiently in dendrites. In contrast, cytoplasmic dynein efficiently navigates both axons and dendrites; in both compartments, dynamic microtubule plus ends enhance dynein-dependent transport. To test the predictions of the optogenetic assay, we examined the contribution of dynein to the motility of an endogenous dendritic cargo and found that dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking. However, inhibition of microtubule dynamics has no effect on BDNF/TrkB motility, suggesting that dendritic kinesin motors may cooperate with dynein to drive the transport of signaling endosomes into the soma. Collectively our data highlight compartment-specific differences in kinesin activity that likely reflect specialized tuning for localized cytoskeletal determinants, whereas dynein activity is less compartment specific but is more responsive to changes in microtubule dynamics.

scholarly journals Dynein Supports Motility of Endoplasmic Reticulum in the FungusUstilago maydis

2002 ◽  
Vol 13 (3) ◽  
pp. 965-977 ◽  
Author(s):  
Roland Wedlich-Söldner ◽  
Irene Schulz ◽  
Anne Straube ◽  
Gero Steinberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Minor Importance ◽  
Dependent Transport

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER inU. maydis.

Microtubule motors and other maps

1990 ◽  
Vol 48 (3) ◽  
pp. 1-1
Author(s):  
Richard B. Vallee
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Structural Components ◽  
Microtubule Associated Proteins ◽  
Microtubule Motors ◽  
Associated Proteins ◽  
The Subject ◽  
Intracellular Motility

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Alexey Bondar ◽  
Olga Rybakova ◽  
Josef Melcr ◽  
Jan Dohnálek ◽  
Petro Khoroshyy ◽  
...  
Keyword(s):  
Biological Systems ◽  
Linear Dichroism ◽  
Quantitative Information ◽  
Living Cells ◽  
Software Tools ◽  
Model Systems ◽  
Membrane Properties ◽  
Polarization Microscopy ◽  
Molecular Processes ◽  
Wide Range

AbstractFluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

scholarly journals E3-13.7 Integral Membrane Proteins Encoded by Human Adenoviruses Alter Epidermal Growth Factor Receptor Trafficking by Interacting Directly with Receptors in Early Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3559-3572 ◽  
Author(s):  
Denise Crooks ◽  
Song Jae Kil ◽  
J. Michael McCaffery ◽  
Cathleen Carlin
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Viral Protein ◽  
Egf Receptor ◽  
Receptor Trafficking ◽  
Model Systems ◽  
Egf Receptors ◽  
Down Regulation ◽  
Human Adenoviruses ◽  
Early Endosomes

Animal cell viruses provide valuable model systems for studying many normal cellular processes, including membrane protein sorting. The focus of this study is an integral membrane protein encoded by the E3 transcription region of human adenoviruses called E3-13.7, which diverts recycling EGF receptors to lysosomes without increasing the rate of receptor internalization or intrinsic receptor tyrosine kinase activity. Although E3-13.7 can be found on the plasma membrane when it is overexpressed, its effect on EGF receptor trafficking suggests that the plasma membrane is not its primary site of action. Using cell fractionation and immunocytochemical experimental approaches, we now report that the viral protein is located predominantly in early endosomes and limiting membranes of endosome-to-lysosome transport intermediates called multivesicular endosomes. We also demonstrate that E3-13.7 physically associates with EGF receptors undergoing E3-13.7–mediated down-regulation in early endosomes. Receptor–viral protein complexes then dissociate, and EGF receptors proceed to lysosomes, where they are degraded, while E3-13.7 is retained in endosomes. We conclude that E3-13.7 is a resident early endocytic protein independent of EGF receptor expression, because it has identical intracellular localization in mouse cells lacking endogenous receptors and cells expressing a human cytomegalovirus-driven receptor cDNA. Finally, we demonstrate that EGF receptor residues 675–697 are required for E3-13.7–mediated down-regulation. Interestingly, this sequence includes a known EGF receptor leucine-based lysosomal sorting signal used during ligand-induced trafficking, which is also conserved in the viral protein. E3-13.7, therefore, provides a novel model system for determining the molecular basis of selective membrane protein transport in the endocytic pathway. Our studies also suggest new paradigms for understanding EGF receptor sorting in endosomes and adenovirus pathogenesis.

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