Abstract
It was recently shown that, in addition to the well-established microtubule-dependent mechanism, fast transport of organelles in squid giant axons also occurs in the presence of actin filaments [Kuznetsov et al., 1992, Nature 356:722-725]. The objectives of this study were to obtain direct evidence of axoplasmic organelle movement on actin filaments and to demonstrate that these organelles are able to move on skeletal muscle actin filaments. Organelles and actin filaments were visualized by video-enhanced contrast differential interference contrast (AVEC-DIC) microscopy and by video intensified fluorescence microscopy. Actin filaments, prepared by polymerization of monomeric actin purified from rabbit skeletal muscle, were stabilized with rhodamine-phalloidin and adsorbed to cover slips. When axoplasm was extruded on these cover slips in the buffer containing cytochalasin B that prevents the formation of endogenous axonal actin filaments, organelles were observed to move at the fast transport rate. Also, axoplasmic organelles were observed to move on bundles of actin filaments that were of sufficient thickness to be detected directly by AVEC-DIC microscopy. The range of average velocities of movement on the muscle actin filaments was not statistically different from that on axonal filaments. The level of motile activity (number of organelles moving/min/field) on the exogenous filaments was less than on endogenous filaments probably due to the entanglement of filaments on the cover slip surface. We also found that calmodulin (CaM) increased the level of motile activity of organelles on actin filaments. In addition, CaM stimulated the movement of elongated membranous organelles that appeared to be tubular elements of smooth endoplasmic reticulum or extensions of prelysosomes. These studies provide the first direct evidence that organelles from higher animal cells such as neurons move on biochemically defined actin filaments.
Highlights
Unconventional myosins represent a diverse group of proteins, many of which are thought to function as organelle motors
In a previous study (Kuznetsov, et al, 1992), we showed that a network of actin filaments formed at the margins of axoplasm after it had been extruded from the squid giant axon and incubated in buffer for 15-30 minutes
Axoplasmic organelles that dissociated from the bulk axoplasm were observed to move along these actin filaments at an average velocity of about 1 μm/second, which is approximately the rate of fast axonal transport
Summary
Unconventional myosins represent a diverse group of proteins (for review see Endow and Titus, 1992; Titus, 1993; Fath and Burgess, 1994), many of which are thought to function as organelle motors. Differential centrifugation has been used to show that myosins cosediment with Golgi membranes in intestinal epithelia (Fath and Burgess, 1993), with small vesicles in Acanthamoeba (Adams and Pollard, 1986) and with contractile vacuoles in Dictyostelium (Zhu and Clarke, 1992). Direct evidence for a role for myosin in the transport of organelles along actin filaments has been more difficult to obtain. Some of the earliest data showing directed movement of organelles along actin filaments came from studies of motility in plant cells and protozoa. The actin-dependent movement of organelles in Chara strongly implicated myosin as the putative motor. Using the Nitella actin cables assay, Adams and Pollard (1986) showed that vesicles from Acanthamoeba moved along actin filaments and this movement could be inhibited by antibodies to Acanthamoeba myosin I. The mouse Dilute and yeast MYO2 proteins, members of the myosin V class of unconventional myosins, have been implicated as organelle motors in studies of organisms that express the mutant forms of these proteins (Mercer et al, 1991; Johnston et al, 1991)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
