New cell biological applications of the laser microbeam technique: the microdissection and skinning of muscle fibers and the perforation and fusion of sarcolemma vesicles.
In a novel approach, the laser microbeam technique was used to selectively perforate the sarcolemma of skeletal muscle fibers, to prepare fragments of myofibrillar bundles of very small dimensions, and to induce fusion of sarcolemma vesicles. Using a highly focused UV laser microbeam with an effective beam diameter of down to 0.5 micron, very small (< 3 microns) myofibrillar fragments with an intact sarcomere striation pattern were obtained. When small amounts of Ca2+ were released in the vicinity of such a fragment by laser-photolysis of the photolabile compound Ca(2+)-nitr-7 the bundle shortened due to the development of calcium-activated force. We also show that very small selected areas from myopathic single muscle cells can be dissected with a precision unmatched by other current techniques. The microbeam was also used to remove very small patches of the sarcolemma of murine skeletal muscle fibers so giving diffusional access to the myoplasmic interior and thus resulting in a "skinning" of the fiber. To ensure that such laser-skinned fiber segments were physiologically intact we determined the Ca(2+)-activated force and caffeine-induced Ca(2+)-release from the sarcoplasmic reticulum. The fibers showed normal characteristics for force production, Ca(2+)-release and uptake by the sarcoplasmic reticulum. To test the effects of the laser microbeam on the muscle membrane directly, we prepared sarcolemma vesicles of skeletal muscle fibers. The vesicles could be selectively perforated with single laser pulses to allow entry of fluorescein isothiocyanate (FITC)-dextran as a fluorescent marker. Adjacent vesicles were caused to fuse by a few pulses at low intensity of the laser microbeam.(ABSTRACT TRUNCATED AT 250 WORDS)
Veigel
C
C
II. Physiologisches Institut, Universität Heidelberg, Germany.
Steubing
R W
RW
Harim
A
A
Weber
C
C
Greulich
K O
KO
Fink
R H
RH
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002458:Cell Fractionation / Q000379:methods; D003911:Dextrans; D004210:Dissection / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D007834:Lasers; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000166:cytology* / Q000648:ultrastructure; D012508:Sarcolemma / Q000378:metabolism / Q000648:ultrastructure*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure
D003911:Dextrans; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium