Differential distribution of vesicular carriers during differentiation and synapse formation

Abstract

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.

A novel AP180-related protein in vesicles that concentrate at acetylcholine receptor clusters

“…Clathrin coated vesicles were isolated and purified from bovine brains as described previously [Bursztajn et al, 1993]. Briefly, brains were homogenized in a Waring blender with three high speed 10 s bursts in two volumes of Buffer A [(0.1 M 2-N-morpholino acid (MES), 0.5 mM MgC1 2 , 1 mM ethylene glycol bis (␤aminoethyl ether)-N,N 1-tetra acetic acid (EGTA), 0.02% NaN3, pH 6.5].…”

“…Because previous studies have shown that coated vesicles contain AChRs and AChE [Bursztajn and Fischbach, 1984;Porter-Jordon et al, 1986;Bursztajn et al, 1991], we collected 3 ml fractions, tested for acetylcholinesterase [Ellman et al, 1961], hexosaminidase [Hall et al, 1978], and NADH cytochrome C reductase [Mahler, 1955] activities, and pooled the fractions enriched in protein and AChE activity but low in hexosaminidase (a marker for lysosomes) and NADH cytochrome C reductase (a marker for mitochondria) activities. Column chromatography revealed two peaks, one of which was enriched in protein and AChE activity [Bursztajn et al, 1993]. After obtaining a single peak from the second chromatography, we centrifuged it for 1 h at 34,000 rpm using a Beckman Type 35 rotor, and assessed the purity of the pellet of coated vesicles so obtained by SDS-polyacrylamide gel electrophoresis [Laemmli, 1970] and electron microscopy [Bursztajn et al, 1993].…”

“…Column chromatography revealed two peaks, one of which was enriched in protein and AChE activity [Bursztajn et al, 1993]. After obtaining a single peak from the second chromatography, we centrifuged it for 1 h at 34,000 rpm using a Beckman Type 35 rotor, and assessed the purity of the pellet of coated vesicles so obtained by SDS-polyacrylamide gel electrophoresis [Laemmli, 1970] and electron microscopy [Bursztajn et al, 1993]. Vesicular membranes were sedimented in a Beckman airfuge at 130,000 g, the pellet was treated with 0.5% saponin and, after washing, the proteins were subjected to 7.5% SDS polyacrylamide gel electrophoresis.…”

“…We and others have shown that some clathrin coated vesicles contain newly synthesized acetylcholine receptors (AChRs) and the enzyme acetylcholinesterase (AChE) [Bursztajn and Fischbach, 1984;Porter-Jordon et al, 1986;Bursztajn et al, 1987;Park and Bursztajn, 1990]. These vesicles, which appear to participate in postsynaptic membrane remodeling and accumulate beneath the nerve muscle contacts, are characterized by reactivity with the epitope for the C172 antibody [Bursztajn et al, 1993].…”

“…To better understand vesicular traffic and postsynaptic membrane remodeling at the neuromuscular synapse, we have generated monoclonal antibodies to the membranes of coated vesicles [Bursztajn et al, 1993]. These vesicles appeared enriched at nerve-muscle contacts, but we did not know whether these regions contained AChR clusters [Bursztajn et al, 1993]. It is well established that in innervated cultures there are nerve-muscle contacts that lack AChR clusters.…”