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Reactivated Melanophore Motility: Differential Regulation and Nucleotide Requirements of Bidirectional Pigment Granule Transport Moshe M

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Reactivated Melanophore Motility: Differential Regulation and Nucleotide Requirements of Bidirectional Pigment Granule Transport Moshe M Reactivated Melanophore Motility: Differential Regulation and Nucleotide Requirements of Bidirectional Pigment Granule Transport Moshe M. Rozdzial and Leah T. Haimo Department of Biology, University of California, Riverside, California 92521 Abstract. To study the molecular basis for organized for aggregation and dispersion were demonstrated by pigment granule transport, procedures were developed observations that aggregation had a lower apparent Km to lyse melanophores of Tilapia mossambica under for ATP than did dispersion and could be initiated at a conditions in which pigment granule movements could lower ATP concentration. Moreover, aggregation could be reactivated. Gentle lysis of the melanophores be initiated by ADP, but only dispersion could be resulted in a permeabilized cell model, which, in the reactivated by the thiophosphate ATP analog, ATPTS. absence of exogenous ATP, could undergo multiple The direction of pigment transport was determined rounds of pigment granule aggregation and dispersion solely by cAMP, since pigment granules undergoing when sequentially challenged with epinephrine and dispersion reaggregated when cAMP was removed, and cAMP. Both directions of transport required ATP, those undergoing aggregation dispersed when cAMP since aggregation or dispersion in melanophores was added. These results provide evidence that pig- depleted of nucleotides could be reactivated only upon ment granule motility may be based on two distinct addition of MgATP or MgATP plus cAMP, respec- mechanisms that are differentially activated and regu- tively. Differences between the nucleotide sensitivities lated to produce bidirectional movements. RGANIZED intracellular particle transport in eukary- onal transport, of latex beads and axoplasmic organelles otic cells is a ubiquitous and functionally important along microtubules. Recent evidence has also indicated the phenomenon. The chmmatophores of vertebrates existence of a presumptive retrograde translocator in axo- provide an excellent model system for studying intracellular plasm 0/ale et al., 1985b). However, the mechanism by motility, which is characterized by the rapid, directed, and which the direction of movement is controlled during axo- cyclic transport of thousands of pigment granules. Aggrega- plasmic transport is unknown. tion, the transport of granules towards the cell center, is of To examine the molecular mechanisms producing bidirec- uniform velocity, while dispersion, or centrifugal pigment tional movements, we have modified the procedures previ- granule transport, is a saltatory process. These movements ously developed to study organized pigment translocation occur as temporally separate events and can be controlled in lysed chromatophores. By permeabilizing melanophores both hormonally and chemically (Bagnara and Hadley, 1973; from the killifish Fundulus, Clark and Rosenbaum (1982, Novales, 1983). Thus, the cellular mechanisms involved in 1984) obtained pigment granule aggregation in vitro and each direction of granule transport can be dissected and demonstrated an ATP requirement for this process in meta- resolved. bolically poisoned cells. In other studies, saltations of pig- Recent studies have revealed that bidirectional organelle ment in lysed erythmphores of the squirrel fish Holocentrus transport can occur along a single microtubule (Hayden and continued in vitro, a characteristic movement of pigment in Allen, 1984; Koonce and Schliwa, 1985; Schnapp et al., the dispersed state in intact cells. This system was used to 1985) and in axoplasm may involve two distinct motors o/ale study the sensitivity of saltatory movements to various ions, et al., 1985a, b). That the force generating motors might be drugs, and antibodies (Stearns and Ochs, 1982). We report associated with the translocating organelles is suggested by here the development of a lysis procedure for melanophores the observation that purified axoplasmic organelles exhibit of the African cichlid, 1Hapia mossambica, in which both bidirectional movements along dynein-free axonemal micro- aggregation and dispersion as well as saltatory movements of tubules (Gilbert et al., 1985). A soluble protein, kinesin, pigment granules, melanosomes, can be reactivated. Results purified from squid and bovine axoplasm (Vale et al., 1985a) of these studies indicate that each of these movements has a and from sea urchin eggs (Scholey et al., 1985), induces the distinct requirement for ATP, that aggregation and dispersion unidirectional movement, corresponding to anterograde ax- are differentially supported by other nucleotides, and that the © The Rockefeller University Press, 0021-9525/86/12/2755/10 $1.00 The Journal of Cell Biology, Volume 103 (No. 6, Pt. 2), Dec. 1986 2755-27,54 2755 direction of melanosome movement is controlled by cAME changes of depletion buffer, which was lysis buffer containing 0.0015 % The results reported here and elsewhere (Rozdzial and digitonin. These nucleotide-depleted cell models only underwent pigment Haimo, 1986) suggest that two distinct mechanisms may be granule aggregation or dispersion upon reactivation with lysis buffer con- taining MgATP or MgATP plus cAMP (reactivation solutions), respec- involved in generating the opposing pigment granule move- tively. Unless otherwise noted, 3 mM ATP (or another nucleotide) and 1 merits. mM cAMP were used in the reactivation solutions. To study pigment gran- A brief account of these results has appeared in abstract ule aggregation, populations of depleted melanophores with dispersed form (Rozdzial and Haimo, 1985). pigment were routinely obtained during lysis into depletion buffer. To ob- tain populations of melanophores with aggregated pigment for studies of melanosome dispersion, cells were first incubated in lysis buffer containing Materials and Methods 100 ~tM epinephrine and then incubated in the depletion buffer. Experimental Materials and Solutions Recording of Experiments The responses of randomly chosen intact or lysed melanophores to drug Live African cichlids, lilapia mossambica, were purchased from Aqua- stimulation or reactivation conditions were observed with differential inter- farms International, Mecca, CA and maintained in freshwater aquaria at ference contrast (DIC) microscopy and recorded with video, photographic, •~25°C. Adenosine thiotriphosphate (ATP3,S) was purchased from Boehr- and photometric equipment. Melanophores were observed and scored for inger Mannheim Biocbemicals, Indianapolis, IN, and bretylium tosylate motility with a Plan 20x DIC objective on a Nikon Optiphot microscope. from Chemical Dynamics Corp., South Plainfield, NJ. All other reagents Video recordings were taken using a Plan 100x oil immersion objective, were purchased from Sigma Chemical Co., St. Louis, MO. Purified IgG a Panasonic NV-8050, half-inch time-lapse recorder (Panasonic Co., Secau- fraction containing rat monoclonal antibodies against yeast tubulin has been cus, NJ), and a camera equipped with a Newvicon tube and circuitry for previously characterized (Kilmartin et al., 1982) and was the generous gift controlling gain, gamma, and black level parameters (67M Series; Dage of Dr. John Kilmartin, Medical Research Council, Cambridge, England. MTI, Inc., Michigan City, IN). Rate measurements of pigment granule Lysis buffer (pH 7.4) consisted of 30 mM Hepes, 10 mM EGTA, 0.5 mM aggregation and dispersion were taken directly from recorded images dis- EDTA, 5 mM MgSOa, 33 mM potassium acetate, 2.5% polyethylene gly- played on a Panasonic WV-5360 monitor that had been calibrated with a col (PEG; 20,000 mol wt), and 0.001% digitonin. Epinephrine, isobutyl- stage micrometer. Real time output was provided by a time-date generator methylxanthine (IBMX), 1 and reserpine were prepared as 100x stock so- (VTG-33; FOR-A Co., West Newton, MA). Photographs were taken with lutions in dimethylsnlfoxide and stored at 40C. Calcium-free goldfish a Nikon FX-35A camera on Plus-X Pan film developed in Rodinol (Agfa- Ringer's (pH 7.4) was composed of 100 mM NaCI, 2.5 mM KC1, 10 mM Gevaert, Inc., Teterboru, NJ). Photometric measurements (see also Clark sodium bicarbonate, and included 1 mM MgC12. Digitonin was freshly and Rosenhaum, 1984) of single cells were taken using the Plan 20× DIC prepared as a 0.4% stock in 50% ethanol (Stearns and Ochs, 1982). objective and a Nikon P1 photometer equipped with a 2-1am pinhole aper- ture and 530-nm filter (on generous loan from A. G. Heinze Co., Inc., Ir- Preparation of Melanophores vine, CA) mounted on the Nikon Optiphot microscope. Changes in light transmittance, recorded and displayed by the photometer digital output, Single scales were removed from fish with forceps and partially cut with were subsequently converted to changes in OD according to Beer's law. The dissecting scissors into several longitudinal sections. To facilitate the re- progressions of pigment granule aggregation and dispersion were related to, moval of the epidermis and expose the underlying melanophores, scales respectively, the observed increases and decreases in OD. These changes were then immersed and agitated in 5 mg/ml collagenase (Type II; Sigma in OD were normalized so that values for aggregation decreased from an Chemical Co.) in Ca++-free Ringer's solution for 1.5 h at room tempera- initial value of 1.0 and dispersion increased from 0. In all figures, n refers ture. The epidermal layer was then removed in Ca++-frec Ringer's. Subse- to the number of cells recorded, and each point represents the average re- quenily, scales were transferred
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