Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique

Helmy, Sherif; Porter-Jordan, K.; Dawidowicz, Eliezar A.; Pilch, Paul F.; Schwartz, Alan L.; Fine, Richard E.

doi:10.1016/0092-8674(86)90471-x

Cited by 49 publications

(23 citation statements)

References 31 publications

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“…In skeletal muscle, a distinct difference between endocytic and exocytic coated vesicles has been reported [Helmy et al, 1986;Porter-Jordan et al, 19861. These investigators used an acetyl-cholinesterase-mediated density shift technique which allowed them t o show that 75% of coated vesicles isolated from embryonic chick skeletal muscle were exocytic [Porter-Jordan et al, 19861.…”

Section: Discussionmentioning

confidence: 98%

Differential distribution of vesicular carriers during differentiation and synapse formation

Bursztajn

Jong

Berman

1993

J of Cellular Biochemistry

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Coated and noncoated vesicles participate in cellular protein transport. Both acetylcholine receptors (AChR) and acetylcholinesterase (AChE) are transported via coated vesicles, some of which accumulate beneath the neuromuscular synapse where AChRs cluster. To investigate the mechanisms by which these proteins are transported during postsynaptic remodeling, we purified coated vesicles from the bovine brain via column chromatography (Sephacryl S-1000) and raised monoclonal antibodies to epitopes of the vesicular membranes enriched in AChE. We assayed for AChE (coated vesicle enriched), hexosaminidase (lysosomal contaminants), NADH cytochrome C reductase (mitochondrial containing), and protein and demonstrated electron microscopically using negative staining that the vesicular fraction contained 95% pure coated vesicles. We then injected coated vesicle fractions and the fractions from which the coat was removed intraperitoneally into mice and obtained three monoclonal antibodies: C-33, C-172, and F-22. On immunoblots of purified vesicles and cultured skeletal muscle, mAb C-33 stained a 180 Kd band and mAb C-172 stained a 100 kd band. MAb F-22 stained 50 kd and 55 kd bands and was not characterized further. Immunofluorescent microscopy with C-33 and C-172 revealed punctate fluorescence whose distribution depends upon the stage of myotube development. Four days after plating, myotubes showed punctate fluorescence throughout the myotube, whereas those stained 8 days after plating showed a punctate perinuclear distribution. Myotubes innervated by ciliary neurons show punctate fluorescence limited to the nuclear periphery and most concentrated around nuclei which line up beneath neuronal processes. This differential vesicular distribution, observed during myotube differentiation and innervation, suggests that these vesicles participate in vesicular membrane traffic.

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Section: Discussionmentioning

confidence: 98%

Differential distribution of vesicular carriers during differentiation and synapse formation

Bursztajn

Jong

Berman

1993

J of Cellular Biochemistry

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“…Although coated vesicles are usually mentioned in the framework of endocytosis, exocytotic coated vesicles are not uncommon (e.g. [33]–[35]). …”

Section: Discussionmentioning

confidence: 99%

3D-Ultrastructure, Functions and Stress Responses of Gastropod (Biomphalaria glabrata) Rhogocytes

et al. 2014

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Rhogocytes are pore cells scattered among the connective tissue of different body parts of gastropods and other molluscs, with great variation in their number, shape and size. They are enveloped by a lamina of extracellular matrix. Their most characteristic feature is the “slit apparatus”, local invaginations of the plasma membrane bridged by cytoplasmic bars, forming slits of ca. 20 nm width. A slit diaphragm creates a molecular sieve with permeation holes of 20×20 nm. In blue-blooded gastropods, rhogocytes synthesize and secrete the respiratory protein hemocyanin, and it has been proposed–though not proven–that in the rare red-blooded snail species they might synthesize and secrete the hemoglobin. However, the cellular secretion pathway for respiratory proteins, and the functional role(s) of the enigmatic rhogocyte slit apparatus are still unclear. Additional functions for rhogocytes have been proposed, notably a role in protein uptake and degradation, and in heavy metal detoxification. Here we provide new structural and functional information on the rhogocytes of the red-blooded freshwater snail Biomphalaria glabrata. By in situ hybridization of mantle tissues, we prove that rhogocytes indeed synthesize hemoglobin. By electron tomography, the first three dimensional (3D) reconstructions of the slit apparatus are provided, showing detail of highly dense material in the cytoplasmic bars close to the slits. By immunogold labelling, we collected evidence that a major component of this material is actin. By genome databank mining, the complete sequence of a B. glabrata nephrin was obtained, and localized to the rhogocytes by immunofluorescence microscopy. The presence of both proteins fit the ultrastructure-based hypothesis that rhogocytes are related to mammalian podocytes and insect nephrocytes. Reactions of the rhogocytes to deprivation of food and cadmium toxification are also documented, and a possible secretion pathway of newly synthesized respiratory proteins through the slit apparatus is discussed.

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“…Coated vesicles were isolated by a modification (14,17) of the method of Blitz et al (1). As previously demonstrated (26), the CVs were at least 95% pure by morphological criteria using both thin section and negative stain EM.…”

Section: Coated Vesicle and L Ysosome Purificationmentioning

confidence: 90%

“…As previously demonstrated (26), the CVs were at least 95% pure by morphological criteria using both thin section and negative stain EM. Over 95% of the cholinesterase activity was demonstrated to be associated with CVs (14). Coated vesicles were then subjected to the cholinesterase density shift protocol as described (14) or fractionated into subpopulations by preparative agarose gel electrophoresis which purifies CVs to apparent homogeneity (16,17,27).…”

Section: Coated Vesicle and L Ysosome Purificationmentioning

confidence: 99%

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

Lemansky¹,

Hasilík²,

Figura³

et al. 1987

The Journal of Cell Biology

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Abstract. The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was ~10-13 %. The implications of these findings are discussed with regard to lysosome trafficking.

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Separation of endocytic from exocytic coated vesicles using a novel cholinesterase mediated density shift technique

Cited by 49 publications

References 31 publications

Differential distribution of vesicular carriers during differentiation and synapse formation

Differential distribution of vesicular carriers during differentiation and synapse formation

3D-Ultrastructure, Functions and Stress Responses of Gastropod (Biomphalaria glabrata) Rhogocytes

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

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