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Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V Stephen L

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Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V Stephen L Regulation of Melanosome Movement in the Cell Cycle by Reversible Association with Myosin V Stephen L. Rogers,* Ryan L. Karcher,* Joseph T. Roland,* Alexander A. Minin,‡ Walter Steffen,§ and Vladimir I. Gelfand* *Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; ‡Institute of Protein Research, Russian Academy of Sciences, Poushchino, Russia 142292; and §Institut für Zellphysiologie und Biosystemtechnik, Mikroskopiezentrum, Universität Rostock, D-18055 Rostock, Germany Abstract. Previously, we have shown that melano- or interphase extract-treated melanosomes. This mitotic somes of Xenopus laevis melanophores are transported inhibition of motility correlated with the dissociation of along both microtubules and actin filaments in a coordi- myosin V from melanosomes, but the activity of soluble nated manner, and that myosin V is bound to purified motor remained unaffected. Furthermore, we find that melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. myosin V heavy chain is highly phosphorylated in Biol. 8:161–164). In the present study, we have demon- metaphase extracts versus interphase extracts. We con- strated that myosin V is the actin-based motor responsi- clude that organelle transport by myosin V is controlled ble for melanosome transport. To examine whether my- by a cell cycle-regulated association of this motor to or- osin V was regulated in a cell cycle-dependent manner, ganelles, and that this binding is likely regulated by purified melanosomes were treated with interphase- or phosphorylation of myosin V during mitosis. metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motil- Key words: myosin • molecular motors • melano- ity of organelles treated with mitotic extract was found phores • melanosomes • regulation to decrease dramatically, as compared with untreated YTOLOGICAL studies of many different types of or- (Bomsel et al., 1990). In neurons, synaptic vesicles move ganelles in animal cells have revealed that their from the cell body to sites of synaptic connections, which C proper spatial and temporal distribution relies may be up to meters away (Amaratunga et al., 1993), while upon transport along elements of the cytoskeleton. The mitochondria are believed to be transported up gradients Golgi apparatus, for example, is restricted to its perinu- of ADP to intracellular sites of high ATP consumption clear position in many differentiated cell types by trans- (Nangaku et al., 1994). Perhaps the most dramatic exam- port to the minus ends of microtubules by cytoplasmic dy- ple of organelle transport occurs during cell division when, nein (Corthesy-Theulaz et al., 1992; Burkhardt et al., during prophase, chromosomes are transported along mi- 1997). In contrast, the steady-state distribution of the ER crotubules to congress at the metaphase plate, and later, in the cell’s periphery is actively mediated by transport to during anaphase, segregate to their respective, opposite the distal, plus ends of microtubules by kinesin (Feiguin et poles (Waters and Salmon, 1997). al., 1994). Other types of organelles are more dynamic and Many years of study have led to the dogma that the ma- must be transported to specific intracellular destinations jority of intracellular transport occurs along micro- to perform their specific tasks. Secretory vesicles travel tubules, driven by the activities of the dynein and kine- from the Golgi apparatus, their point of origin, to the sin superfamilies of motor proteins. However, in recent plasma membrane for exocytosis (Lafont and Simons, years, this assumption has been challenged by the demon- 1996). Endosomes are transported centripetally from the stration that some organelles can move along filamentous cell’s periphery to its interior for subsequent processing actin (Adams and Pollard, 1986; Kuznetzov et al., 1992; Bearer et al., 1993; Mermall et al., 1994; Morris and Hol- lenbeck, 1995; Krendel et al., 1998). Compelling evidence Address correspondence to Dr. Vladimir I. Gelfand, Department of Cell that a single type of organelle can be transported along and Structural Biology, University of Illinois at Urbana-Champaign, B107 both microtubules and actin filaments has resulted from Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Ur- bana, IL 61801. Tel.: (217) 333-5972. Fax: (217) 333-5982. E-mail: vgelfand the study of pigment granule transport in melanophores, a @uiuc.edu model system for the study of intracellular transport. The The Rockefeller University Press, 0021-9525/99/09/1265/11 $5.00 The Journal of Cell Biology, Volume 146, Number 6, September 20, 1999 1265–1275 http://www.jcb.org 1265 role of these cells, present in the dermal layers of fish tion. The melanosomes present in these cells may be puri- and amphibians, is to cyclically aggregate pigmented or- fied rapidly and in large quantities, and have been shown ganelles, termed melanosomes, to the center of the cell or to exhibit both microtubule- and actin-based motility in disperse them throughout the cytoplasm to effect color vitro. Treatment of isolated melanosomes with Xenopus changes in the animal’s skin (Haimo and Thaler, 1994). In egg extracts arrested either in metaphase or interphase al- Xenopus laevis melanophores, pigment transport is regu- lows the study of cell cycle-dependent regulation of the lated by hormone-induced modulation of intracellular microtubule- and actin-based motors present on these or- cAMP levels: melanocyte-stimulating hormone (MSH)1 ganelles. In this study, we have demonstrated that myosin triggers dispersion by upregulation of cAMP production, V is the motor responsible for actin-based transport of while melatonin induces pigment aggregation by downreg- melanosomes in Xenopus melanophores through the use ulating cAMP levels (Daniolos et al., 1990). This hor- of a dominant-negative myosin V construct and by immu- mone-induced organelle transport is regulated by antago- nofluorescent localization of the motor to melanosomes. nistic cycles of kinase and phosphatase activities (Reilein We then used our system to study the regulation of myo- et al., 1998). Until recently, it was believed that melano- sin V during mitosis. Treatment of melanosomes with somes were exclusively carried along the cells’ radially or- metaphase, but not interphase, extracts resulted in a dra- ganized microtubule cytoskeleton with a kinesin-related matic decrease in vitro motility. This decreased motility protein, kinesin-II, transporting pigment to the microtu- was due to dissociation of myosin V from pigment gran- bule plus ends during dispersion and dynein moving them ules and not due to inhibition of its motor activity. The to the minus ends during aggregation (Nilson and Wallin, myosin V heavy chain exhibited a substantial increase in 1997; Tuma et al., 1998). It is now clear, however, that an- phosphate incorporation in mitotic extracts, compared other, actin-based component also contributes to pigment with interphase extracts, implicating phosphorylation of transport in melanophores. Upon disruption of the micro- myosin V as the regulatory mechanism. To our knowl- tubule cytoskeleton, melanosomes exhibit short, shuttling edge, this is the first study documenting a molecular movements that halt in the presence of actin-depolymeriz- mechanism for the cell cycle-mediated regulation of ac- ing drugs (Rodionov et al., 1998). Furthermore, we have tin-based organelle transport. demonstrated that purified melanosomes can move along actin filaments in vitro and that the actin-based motor, my- osin V, is associated with these organelles (Rogers and Materials and Methods Gelfand, 1998). Similar findings of coordinated actin- and microtubule-based transport were also reported for me- Melanophore Cell Culture and Transfection lanosomes in cultured mouse melanocytes (Wu et al., Immortalized Xenopus melanophores were cultured as described previ- 1998a). ously (Rogers et al., 1997). Immunofluorescent localization of myosin V was performed using a clonal nonpigmented cell line, clone 47, or gray The mitotic cell is confronted with the important task of cells, derived from the original melanophore cell line (Daniolos et al., ensuring that both daughter cells receive their appropriate 1990). Melanophores containing a lower melanin content were selected allotment of each organelle type (Warren, 1993; Warren by freezing the original cell line in 95% FCS and 5% DMSO, according and Wickner, 1996; Shima et al., 1998). Since the inter- to standard protocols. Approximately 5% of the cells survived thawing phase distributions of many organelles rely upon the activ- and reculturing, many of them possessing large vesicles containing small (z0.2 mm) particles of melanin. This cycle of freezing and thawing was re- ities of motor proteins, it stands to reason that their segre- peated once again and pigment-deficient cells were cloned twice on 10-cm gation during mitosis must be accompanied by modulation tissue culture plates using the cloning ring technique. A morphologically of the activities of associated motors. At present, the Allan stable clone was selected and expanded. Over 50% of the cells in this pop- and Vale laboratories have performed the only studies di- ulation contained numerous unmelanized vesicles z1-mm diam. These vesicles responded to hormone treatment by aggregating and dispersing as rectly examining this topic. Using Xenopus frog egg ex- normal melanosomes and were, therefore, considered pigment-free
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