Diffusive and nondiffusive proteins in vivo.

Paine, Philip L.

doi:10.1083/jcb.99.1.188s

Cited by 56 publications

(34 citation statements)

References 37 publications

(36 reference statements)

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“…This type of multienzyme association has been described for the glycolytic enzyme pathway (31) and may be a common cellular organizational scheme. In fact, it has recently become clear that most proteins within the cell are not completely free to diffuse throughout the cytosol (32) and that they behave as if the viscosity they experience is many times higher than that expected in an aqueous compartment (33). This implies that "cytosolic" proteins are commonly subject to noncovalent associations or barriers within the cell that impair their diffusion and are probably important for their optimal function.…”

Section: Resultsmentioning

confidence: 99%

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Kelner¹,

Morita²,

Rossen³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosie,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14. 16.2] and phenylethanolamine N-methyltransferase (PMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1. 1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffln cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (S jM), (ii) a "digitonin-sensitive" cytoplasnic protein pool, which required higher concentrations of digitonin for release (10 ,uM) and included TyrOHase and PMTase, and (iii) the chromaffln granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 IAM than at 40 jaM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase eftlux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromffin granule.In epinephrine-forming cells of the adrenal medulla, the catecholamine biosynthetic pathway is composed of four enzymes, tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], aromatic amino acid decarboxylase (aromatic L-amino acid carboxy-lyase, EC 4.1.1.28), dopamine ,B-hydroxylase [dopamine l3-monooxygenase; 3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (P-hydroxylating), EC 1.14.17.1] and phenylethanolamine N-methyltransferase (PMTase; Sadenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28). Dopamine o-hydroxylase is localized in the interior of the chromaffin granule and on the internal face of the granule membrane (1), while TyrOHase, aromatic amino acid decarboxylase, and PMTase are generally considered to be "soluble" cytoplasmic enzymes and ate synthesized on free ribosomes (2-4). However, morphological and subcellular fractionation studies suggest the possible particulate localization of PMTase and TyrOHase, perhaps on the surface of the chromaffmi granule. By immunocytochemistry, both PMTase and TyrOHase have been seen to be associated with chromaffin granules (5-9), and one report shows TyrOHase to be localized on microtubules (8). In purification procedur...

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Section: Resultsmentioning

confidence: 99%

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Kelner¹,

Morita²,

Rossen³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…These studies have proposed the following about motile fibroblasts: The cytoplasm, instead of showing fluid properties alone, appears to be a highly crosslinked gel of cytoskeletal fibers, as evidenced by electron microscopy (EM) [Porter, 1976;Heuser and Kirschner, 19801 and the fact that the diffusion of particles larger than a typical 100 kDa protein is restricted, as measured by fluorescence photobleaching recovery [Jacobson and Wojcieszyn, 1984;Paine, 1984;Luby-Phelps et al, 19861. This suggests that the cell cytoplasm does not simply flow forward as would a fluid during locomotion.…”

Section: Introductionmentioning

confidence: 98%

Human neutrophil motility: Time‐dependent three‐dimensional shape and granule diffusion

Felder

Kam

1994

Cell Motil. Cytoskeleton

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The locomotion of human polymorphonuclear leukocytes (PMNs) was studied with two complementary methods: Three-dimensional shapes were reconstructed from time series of optical sectioning microscopy using differential interference contrast (DIC) optics, and the diffusion of cytoplasm granules within individual cells was measured using quasielastic laser light scattering (QELS). The three-dimensional cell edges outlined in the optical sections were analyzed qualitatively in time-lapse film strips and quantitatively from morphometry. The fastest locomotion occurred in chemotactic gradient with cell velocity that oscillated between 10 and 30 microns/min with a period of 50-55 seconds. Within the periodic bursts of speed, a fibroblast-like locomotory cycle was observed, with leading lamella extended and contacts formed with the substrate surface, followed by rapid motion of the cell body and nucleus over the immobile contacts. Consistent with this apparent staged motion, correlation analysis revealed a phase lag of 2-3 seconds in velocities between the bottom (ventral) and the top layers of the cell. In addition there was a tendency to a lower cell profile at times of higher velocity. The diffusion of natural cytoplasmic granules within resting PMNs was not affected by cytoskeleton disrupting drugs. During the stage of most rapid motion, when cytoplasmic streaming could be seen, diffusion of the granules decreased two- to 2.5-fold, and then returned to resting levels. These observations suggest that PMN locomotion consists of extensions near the surface to form forward contacts and then stiffening or possibly contraction of the cytoskeleton when the body of the cell is moved forward. Three-dimensional movies of PMN cells are included in the video supplement.

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“…Novel methods to study the diffusion and distribution of molecules within large, living cells have been developed with the rationale that studies of the motion of macromolecules in the cytoplasm will provide information concerning the structure and apparent viscosity of this compartment (8)(9)(10)(11). Recently, the photobleaching techniques have been used to measure the diffusion of exogenously injected foreign and native macromolecules within the cytoplasm of living cells (12)(13)(14)(15).…”

mentioning

confidence: 99%

The translational mobility of substances within the cytoplasmic matrix.

Jacobson¹,

Wojcieszyn²

1984

Proc. Natl. Acad. Sci. U.S.A.

139

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The translational mobility of fluorescent-labeled molecules injected into the cytoplasm of living cells can be measured by the fluorescence recovery after photobleaching (FRAP) technique. In the fibroblast cytoplasm, the diffusion coefficients, D, of test macromolecules ranging in molecular weight from 12,000 to 440,000 are about 10-8 cm2/sec and exhibit almost no dependence on molecular weight. FRAP experiments also showed that macromolecular diffusion within Sepharose beads having an effective pore size smaller than the "microtrabecular lattice" is only slightly retarded compared to buffer values-in contrast to the marked retardation measured in the cytoplasm. This leads to the conclusion that diffusion in the cytomatrix is dominated not by steric effects but rather by binding of the diffusing species to elements of the cytomatrix. These diffusion rates were difficult to modulate; cytochalasin, colchicine (except at 50C), and taxol treatments had little effect. The diffusion rates were not dependent on cellular energy metabolism. However, hypotonic treatment increased the D for bovine serum albumin by nearly 2-fold, whereas hypertonic treatment halved D. Withdrawing the free water from the cell by using 44% polyethylene glycol treatment stopped the translational mobility of the test molecules. A survey of the recent literature is presented, which shows that major differences in the cytomatrix of different cell types exist with respect to the translational diffusion of injected probes.Finally, the spectrum of cytoplasmic translational mobilities ranging from small molecules to organelles is discussed.

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Diffusive and nondiffusive proteins in vivo.

Cited by 56 publications

References 37 publications

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Human neutrophil motility: Time‐dependent three‐dimensional shape and granule diffusion

The translational mobility of substances within the cytoplasmic matrix.

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