scholarly journals Microtubule-based Endoplasmic Reticulum Motility in Xenopus laevis: Activation of Membrane-associated Kinesin during Development

1999 ◽  
Vol 10 (6) ◽  
pp. 1909-1922 ◽  
Author(s):  
Jon D. Lane ◽  
Victoria J. Allan
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Differential Interference Contrast Microscopy ◽  
Animal Cells ◽  
Microtubule Motor ◽  
Directed Motility ◽  
Conventional Kinesin

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts ofXenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopusegg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development inXenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute ∼19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower (∼0.4 μm/s) than minus end-directed motility (∼1.3 μm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

scholarly journals Dynactin is required for bidirectional organelle transport

2003 ◽  
Vol 160 (3) ◽  
pp. 297-301 ◽  
Author(s):  
Sean W. Deacon ◽  
Anna S. Serpinskaya ◽  
Patricia S. Vaughan ◽  
Monica Lopez Fanarraga ◽  
Isabelle Vernos ◽  
...  
Keyword(s):  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Opposite Polarity ◽  
Model System ◽  
Organelle Transport ◽  
Transport Activity ◽  
Microtubule Motors ◽  
Microtubule Motor ◽  
Dynactin Complex ◽  
Binding Subunit

Kinesin II is a heterotrimeric plus end–directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II–mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II–associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530–793 of XKAP and aa 600–811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

scholarly journals Increased Cellular Uptake of Polyunsaturated Fatty Acids and Phytosterols from Natural Micellar Oil

Nutrients ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 150 ◽  
Author(s):  
Clemens Röhrl ◽  
Flora Stübl ◽  
Martin Maier ◽  
Bettina Schwarzinger ◽  
Clemens Schwarzinger ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Cell Types ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Target Cells ◽  
Hydrophobic Compounds ◽  
Lipid Transporters ◽  
Algae Oil

The transport of hydrophobic compounds to recipient cells is a critical step in nutrient supplementation. Here, we tested the effect of phospholipid-based emulsification on the uptake of hydrophobic compounds into various tissue culture cell lines. In particular, the uptake of ω-3 fatty acids from micellar or nonmicellar algae oil into cell models for enterocytes, epithelial cells, and adipocytes was tested. Micellization of algae oil did not result in adverse effects on cell viability in the target cells. In general, both micellar and nonmicellar oil increased intracellular docosahexaenoic acid (DHA) levels. However, micellar oil was more effective in terms of augmenting the intracellular levels of total polyunsaturated fatty acids (PUFAs) than nonmicellar oil. These effects were rather conserved throughout the cells tested, indicating that fatty acids from micellar oils are enriched by mechanisms independent of lipases or lipid transporters. Importantly, the positive effect of emulsification was not restricted to the uptake of fatty acids. Instead, the uptake of phytosterols from phytogenic oils into target cells also increased after micellization. Taken together, phospholipid-based emulsification is a straightforward, effective, and safe approach to delivering hydrophobic nutrients, such as fatty acids or phytosterols, to a variety of cell types in vitro. It is proposed that this method of emulsification is suitable for the effective supplementation of numerous hydrophobic nutrients.

scholarly journals Tissue-culture Cell fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients

1979 ◽  
Vol 182 (1) ◽  
pp. 165-171
Author(s):  
J M Graham ◽  
K H M Coffey
Keyword(s):  
Tissue Culture ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Cell Fractionation ◽  
Early Passage ◽  
Late Passage ◽  
Passage Cells ◽  
Zonal Rotor

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.

scholarly journals Apoptotic Cleavage of Cytoplasmic Dynein Intermediate Chain and P150GluedStops Dynein-Dependent Membrane Motility

2001 ◽  
Vol 153 (7) ◽  
pp. 1415-1426 ◽  
Author(s):  
Jon D. Lane ◽  
Maïlys A.S. Vergnolle ◽  
Philip G. Woodman ◽  
Victoria J. Allan
Keyword(s):  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Animal Cells ◽  
Dynein Intermediate Chain ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Dynactin Complex ◽  
Intermediate Chains

Cytoplasmic dynein is the major minus end–directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150Glued. We have found that both CD-IC and p150Glued are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH2-terminal p150Glued binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150Glued in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein–driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein–dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

scholarly journals Tissue-culture cell fractionation. Fractionation of cellular membranes from 125I/lactoperoxidase-labelled Lettrée cells homogenized by bicarbonate-induced lysis: resolution of membranes by zonal centrifugation and in sucrose and metrizamide gradients

1979 ◽  
Vol 182 (1) ◽  
pp. 173-180 ◽  
Author(s):  
J M Graham ◽  
K H M Coffey
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Cell Fractionation ◽  
Rate Gradient ◽  
Nadph Cytochrome C Reductase ◽  
Zonal Rotor

1. Lettrée cells were grown intraperitoneally in MF-1 mice and labelled extrinsically by the 125I/lactoperoxidase technique. 2. The cells were swollen in 1 mM-NaHCO3 and disrupted in a Dounce homogenizer. 3. Crude fractions of endoplasmic reticulum, plasma membrane and mitochondria were separated from a post-nuclear supernatant by sedimentation-rate gradient centrifugation in a BXIV zonal rotor. 4. Further resolution of these membranes was carried out in isopycnic sucrose gradients. 5. Bands of material from the latter were subfractionated in gradients of metrizamide. Some very pure subfractions of plasma membrane and endoplasmic reticulum were obtained. In addition, one subfraction containing 125I and NADPH-cytochrome c reductase but no Na++K+-stimulated adenosine triphosphatase and another containing these two enzymes but no 125I were resolved.

scholarly journals Pac1/LIS1 promotes an uninhibited conformation of dynein that coordinates its localization and activity

2019 ◽  
Author(s):  
Matthew G. Marzo ◽  
Jacqueline M. Griswold ◽  
Steven M. Markus
Keyword(s):  
Single Molecule ◽  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Extrinsic Factors ◽  
Temporal Precision ◽  
Negative Stain ◽  
Microtubule Motor ◽  
And Function ◽  
High Degree ◽  
In Cells

ABSTRACTCytoplasmic dynein is a minus end-directed microtubule motor that transports myriad cargos in various cell types and contexts. How dynein is regulated to perform all these activities with a high degree of spatial and temporal precision is unclear. Recent studies have revealed that human dynein-1 and dynein-2 can be regulated by a mechanism of autoinhibition, whereby intermolecular contacts limit motor activity. Whether this autoinhibitory mechanism is conserved throughout evolution, whether it can be affected by extrinsic factors, and its precise role in regulating cellular dynein activity remain unknown. Here, we use a combination of negative stain EM, single molecule motility assays, genetic, and cell biological techniques to show that the autoinhibitory conformation is conserved in budding yeast, and it plays an important role in coordinating dynein localization and function in cells. Moreover, we find that the Lissencephaly-related protein, LIS1 (Pac1 in yeast) plays an important role in regulating this autoinhibitory conformation of dynein. Specifically, our studies demonstrate that rather than inhibiting dynein motility, Pac1/LIS1 promotes dynein activity by stabilizing the uninhibited conformation, which ensures appropriate localization and activity of dynein in cells.

Regulation of processive motion and microtubule localization of cytoplasmic dynein

2015 ◽  
Vol 43 (1) ◽  
pp. 48-57 ◽  
Author(s):  
Rupam Jha ◽  
Thomas Surrey
Keyword(s):  
Present Article ◽  
Force Production ◽  
Maximum Velocity ◽  
Cell Types ◽  
Cytoplasmic Dynein ◽  
Present Review ◽  
Microtubule Motor ◽  
Different Cell Types

The cytoplasmic dynein complex is the major minus-end-directed microtubule motor. Although its directionality is evolutionary well conserved, differences exist among cytoplasmic dyneins from different species in their stepping behaviour, maximum velocity and force production. Recent experiments also suggest differences in processivity regulation. In the present article, we give an overview of dynein's motile properties, with a special emphasis on processivity and its regulation. Furthermore, we summarize recent findings of different pathways for microtubule plus-end loading of dynein. The present review highlights how distinct functions in different cell types or organisms appear to require different mechanochemical dynein properties and localization pathways.

A Method for Electron Microscopic Study of Tissue Culture Cell Monolayers Grown in Tubes

1967 ◽  
Vol 25 ◽  
pp. 132-133
Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Light Microscopy ◽  
Cell Sheet ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Electron Microscopic ◽  
Cell Sheets ◽  
Morphological Alterations ◽  
Culture Cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

Prevalence of the pit and antipit in the genus Glycine

1987 ◽  
Vol 45 ◽  
pp. 986-987
Author(s):  
R. W. Yaklich ◽  
E. L. Vigil ◽  
W. P. Wergin
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Seed Coat ◽  
Cell Types ◽  
Abaxial Surface ◽  
Legume Seed ◽  
Initial Report ◽  
Cone Cells ◽  
Glycine Max L

The legume seed coat is the site of sucrose unloading and the metabolism of imported ureides and synthesis of amino acids for the developing embryo. The cell types directly responsible for these functions in the seed coat are not known. We recently described a convex layer of tissue on the inside surface of the soybean (Glycine max L. Merr.) seed coat that was termed “antipit” because it was in direct opposition to the concave pit on the abaxial surface of the cotyledon. Cone cells of the antipit contained numerous hypertrophied Golgi apparatus and laminated rough endoplasmic reticulum common to actively secreting cells. The initial report by Dzikowski (1936) described the morphology of the pit and antipit in G. max and found these structures in only 68 of the 169 seed accessions examined.

scholarly journals Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

2016 ◽  
Vol 113 (34) ◽  
pp. E4995-E5004 ◽  
Author(s):  
Wen Lu ◽  
Michael Winding ◽  
Margot Lakonishok ◽  
Jill Wildonger ◽  
Vladimir I. Gelfand
Keyword(s):  
Cytoplasmic Streaming ◽  
Cell Types ◽  
Nurse Cells ◽  
Wild Type ◽  
Actin Cortex ◽  
Microtubule Motor ◽  
Multiple Cell ◽  
Cytoplasmic Microtubules ◽  
Two Populations

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

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