High-resolution imaging of protein phosphorylation in squid axons and synapse by electron energy loss spectroscopy.

Abstract

We demonstrate that clusters of phosphorus atoms can be detected in energy loss specuoscopic images (ESI) o f c y t~l etal proteins of squid axons. In series of images taken at four energy windows below and three windows above the phosphorus P-L2,3 ionization edge, signal-to-background intensity differences were analyzed by videodensitometry. A distinct increase of relative intensities was recorded above the phosphorus edge in neurofilaments of the peripheral giant axon and in those of the presynaptic terminal. A high level of n e w o f h e m phosphorylation in the peripheral axon sup

Active Polysomes Are Present in the Large Presynaptic Endings of the Synaptosomal Fraction from Squid Brain

“…Because ribosomes contain several thousand phosphate groups in a limited volume, they may be identified as bright signals of 25 nm diameter on a dark background (Korn et al, 1983). These signals may be distinguished from those emitted by phosphoproteins on the basis of their spectral properties (Martin et al, 1993). Using the ESI method, polysomes have been detected in postsynaptic and distal regions of the squid giant axon (Martin et al, 1989;Giuditta et al, 1991) and more recently in the cortical regions of the goldfish Mauthner axon (Koenig and Martin, 1996).…”

Videodensitometric analysis of electron spectroscopic micrographs — a tool for detection of biologically relevant elements with high resolution

“…S L2,3 ¼ sulphur L 2,3 edge. exposure time is reasonable for specimens consisting of large background areas and small structural dots, as is the case with samples of squid axons examined previously (Martin et al, 1993). The assumption is, however, not valid in some of the specimens investigated here because they consisted of relatively large structures instead of dots.…”

“…Applying a videodensitometric method, the visualization of phosphorylation sites in cytoskeletal proteins of squid axons was possible (Martin et al, 1993). Some peculiarities of this method will be discussed here further to verify the reliability of the results.…”

“…Since the axoplasmic signals examined previously (Martin et al, 1993) were too small for EELS, neurofilament-enriched pellets with accumulation of signals were used for EELS analysis. Neurofilaments were extracted from axoplasmic samples as described (Tytell et al, 1988).…”

“…They were processed in a 512 × 512 matrix with 256 grey levels for each pixel. A specially designed computer program was used to measure the optical densities of the relevant structures and of the background surrounding them (for further details, see Martin et al, 1993). The optical densities of the relevant structures were obtained as background-subtracted net densities, as described in Section 2.…”