Taking the A-train: actin-based force generators and organelle targeting

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Taking the A-train: actin-based force generators and organelle targetingq Kammy L. Fehrenbacher, Istvan R. Boldogh and Liza A. Pon Department of Anatomy and Cell Biology, Columbia University, 630 West 168th Street – P&S 12 – 425, New York, NY 10032, USA

The actin-driven process of cytoplasmic streaming in plant cells is widely believed to be the earliest documented example of cytoskeleton-driven organelle movement. In the decades since these seminal findings, two mechanisms of actin-based intracellular movement have been identified in multiple cell types: one is myosin dependent and the other is dependent upon the Arp2/3 complex for actin nucleation and polymerization. Here, we describe mechanisms of force generation and directed movement that use the actin cytoskeleton, as well as those that target actin-dependent force generators to different subcellular compartments. In plant cells such as Chara and Nitella, which measure 3 mm– 10 cm in length, the transport of nutrients and metabolites along the length of the cell by simple diffusion might require days or weeks. Such large cells have developed an alternative mechanism to ensure effective cytoplasmic mixing. In this process, known as cytoplasmic streaming, organelles and vesicles undergo vigorous movement around a large central vacuole at velocities of 30 – 100 mm sec21. A role for the actin cytoskeleton in this process emerged from various studies: (a) Nagai and Rebhun [1] showed that bundles of actin filaments aligned along the axis in the cortical regions of Nitella cells; (b) Bradley [2] showed that cytoplasmic streaming could be inhibited by destabilization of F-actin with cytochalasin;

and (c) Kachar and Reese [3,4] observed unidirectional movement of organelles along actin bundles in Chara and in extruded cytoplasm from Chara. ater studies indicated that actin-dependent intracellular movement was not restricted to plants. Using mainly pharmacological techniques, such as cytochalasin treatment and light microscopy to visualize organelles and the cytoskeleton, it was firmly established that actin-filamentdependent movement of membrane-delimited vesicles occurred in the growth cone lamellipodia of cultured superior cervical ganglion neurons and extruded squid axoplasm [5–7]. The polarity, ATP dependence and drug sensitivity of organelle movement along actin cables of Chara and other organisms was similar to that of heavy meromyosin-driven bead movement and myosin-driven Acanthamoeba organelle movement along actin filaments [8,9]. Consistent with this, proteins that cross-reacted with myosin antibodies or those that had myosin-like activity were identified in Nitella [10 –12]. To date, at least 18 myosin types have been identified (reviewed by Schliwa and Woehlke [13]). One particular class of unconventional myosins (type V) has been implicated in actin-dependent organelle movement in unicellular and metazoan organisms (Table 1 and [7,9,14–18]). Type V myosin, purified from chick brain, is a plus-end directed actin-dependent motor consisting of 2 heavy chains. Each heavy chain contains an N-terminal motor or head domain, a neck region that

Table 1. Organelle transport roles for myosin V Organism

Organelle/cargo

Receptor

Myosin

Refs

Saccharomyces cerevisiae

Peroxisomes Late Golgi (subset) Secretory vesicles Vacuole Melanosomes Dense brain vesicles Smooth ER vesicles Plasma membrane Recycling vesicles (Rab11a associated) Plasma membrane Recycling vesicles (transferrin receptor-containing)

Unknown Unknown Sec4p/Sec2p Vac8p/Vac17p Rab27a/melanophilin Unknown Unknown Rab11a/Rab11-FIP

V V V V Va Va Va Vb

[32] [33] [61,76] [73] [21,64– 65,67–70] [77] [7,78] [62,79– 81]

Rab11a/Rab11-FIP Rab8

Vb Vc

[62,79– 81] [63]

Human/mouse melanocytes Rat/chicken Squid Canine MDCK HeLa

Abbreviations: ER, Endoplasmic Reticulum; MDCK, Madin-Darby Canine Kidney; Rab11-FIP, Rab11-Family Interacting Protein.

q The title is a reference to one of the best-known pieces from the Duke Ellington Orchestra, “Take the A Train”. The A train is the longest-running express train in New York City. It brought Billy Strayhorn, the composer and writer of the song, to Duke Ellington’s band in Harlem in 1940 and brings us to the lab every day. Corresponding author: Liza A. Pon ([email protected]).

http://ticb.trends.com 0962-8924/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0962-8924(03)00174-0

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binds to six calmodulin light chains and a tail that contains a proximal coiled-coil and a distal globular domain [19– 22]. In this review, we discuss myosin-dependent and myosin-independent mechanisms for actin-based intracellular movement and the processes underlying targeting of various actin-dependent force generators to their cargo. Myosin-V-driven organelle movement A role for type V myosins in organelle and particle movement in budding yeast is suggested by phenotypic analysis of loss-of-function myosin mutants. There are two type V myosins in budding yeast, Myo2p and Myo4p. Deletion of the gene encoding Myo4p results in defects in movement of the mRNA encoding Ash1p (a protein required for mating-type switching) from mother to daughter cells during cell division [23,24]. Myo2p is essential and accumulates in the bud tip. Mutation of this protein results in defects in transport of secretory vesicles, the vacuole, peroxisomes, late Golgi elements and components involved in mitotic spindle orientation from mother cells to buds along actin cables [25 –33]. Similar approaches have revealed a role for type V myosins in other intracellular particle movements. For example, antibodies against myosin V proteins detected this motor on endoplasmic reticulum (ER) isolated from squid axoplasm and inhibited actin-based ER movement in extruded squid axoplasm [7]. A similar mechanism underlies transfer of pigment-containing melanosomes from melanocytes to keratinocytes. During intracellular transport, melanosomes initially undergo microtubuledependent movement through melanocyte dendrites. Thereafter, they are transferred to the actin cytoskeleton for movement through the actin-rich cortex of the dendrite. In dilute mice, which bear a mutation in the myosin Va gene, melanosomes undergo microtubuledependent movement; however, they fail to undergo actin-dependent movement in the dendritic cortex. This, in turn, prevents melanosome accumulation in the periphery of dendrites and produces the characteristic lack of pigmentation in dilute mice [34– 37,21]. The model that emerged from these studies is one in which type V myosin motors utilize tail subdomains to bind to their organelle passenger. Sequences in the myosin motor domain bind to the lateral surface of actin filaments in the absence of ATP. As a result of ATP hydrolysis, a conformational change in the globular motor domain is translated into movement through the tilting of the myosin lever arm relative to the myosin globular head. Repetitive rounds of these interactions lead to processive ‘walking’ of myosin and their organelle passengers towards the fast-growing plus, or barbed, ends of actin filaments. Arp2/3-complex-driven organelle movement A great deal of information for the mechanism of actin polymerization-based movement comes from studies of pathogens, including Listeria, Shigella and vaccinia virus (reviewed in [38]). A common feature of these organisms is that, after infection, a ‘comet tail’ consisting of F-actin and various actin-binding proteins drives their movement within the cytoplasm of the infected cell and/or at the plasma membrane for transmission from one cell to a http://ticb.trends.com

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neighboring cell. Electron microscopy of this actin tail revealed that the constituent actin filaments are oriented with their barbed, fast-growing ends towards the surface of the motile bacterium [39,40]. Experiments also showed that photo-activated marks, when introduced into a short segment of Listeria tail in infected cells, remained stationary while the bacterium moved [41]. These observations, which indicate that actin polymerization occurs at the interface between the bacteria and the actin tail, were later confirmed by fluorescent speckle microscopy studies [42]. The Arp2/3 complex is the major factor in nucleation of actin assembly in the actin comet tail of motile bacteria and at many sites of actin polymerization [43]. This evolutionarily conserved seven-subunit complex is regulated by a variety of nucleation-promoting factors (NPFs). Like the best-characterized NPF, WASp, all NPFs contain a conserved Arp2/3-binding sequence, consisting of a short stretch of basic and acidic domains, that is necessary for Arp2/3 complex activation (reviewed in [44]). In its activated state, the Arp2/3 complex binds to actin filaments at their pointed, slow-growing end, allowing for addition of actin monomers at the barbed, fast growing end [45]. Arp2/3 complex can also bind to an existing filament to create and stabilize filament branches [46]. These activities of Arp2/3 lead to assembly of a higherorder dendritic array at the membrane surface, eventually leading to a branched network of actin filaments in vivo and in vitro [47,48]. Recent studies indicate that Arp2/3 complex and actin polymerization drive movement of endogenous organelles and/or particles. Actin comet tails have been detected in association with motile, endogenous vesicles, such as pinosomes, endosomes and phagosomes [49– 52]. Consistent with this, the NPF N-WASp localizes to endosomes and is required for endosome movement driven by actin comet tails in Xenopus extracts and budding yeast [50,53]. Several different signal-transduction proteins have been implicated in the recruitment of N-WASp to endosomes (Fig. 1). These include protein kinase C and Cdc42 in Xenopus extracts [50], and phosphatidylinositol-4-phosphate 5-kinase, Nck (an SH3– SH2 adaptor protein) and WIP (the WASp-interacting protein) in cultured fibroblasts [54,55]. Work from other laboratories demonstrates that other organelles and particles use actin polymerization as a vehicle for force generation. Arp2/3 complex has been detected on the yeast vacuole [56]. Moreover, Boldogh et al. [57] provided evidence for a role for the Arp2/3 complex and actin polymerization in mitochondrial movement and inheritance in yeast. First, mitochondrial movement requires constant actin assembly and disassembly and is impaired by an agent that perturbs actin dynamics. Second, Arp2/3 complex subunits colocalize with mitochondria in intact cells and are tightly associated with the surface of isolated yeast mitochondria. Lastly, mutations in Arp2/3 complex subunits inhibit mitochondrial movement, yet have no obvious effect on colocalization of mitochondria with actin cables [57]. These findings indicate that the Arp2/3 complex is associated with yeast mitochondria and that Arp2/3-complex-driven actin

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F-actin in the actin comet tail of mitochondria to actin cables. Together, these activities allow yeast mitochondria to link the force-generating ability of actin polymerization to the machinery that establishes cell polarity during mitochondrial inheritance.

(a)

Xenopus endosome

N-WASP

Cdc42 targeting factor

Cdc42

Actin filament Arp2/3 complex Actin monomer

(b)

P

P

Fibroblast endosome P

Tyr-phosphorylated organelle-specific protein

Nck WIP N-WASP

Arp2/3 complex Actin monomer Actin filament

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Fig. 1. Two models for recruitment of the actin assembly machinery to endogenous vesicles. (a) Activation of Xenopus extracts by phorbol ester results in the assembly of dynamic actin tails on endosomal and lysosomal vesicles. N-WASP is recruited to the vesicles that are associated with actin comets. This recruitment requires Cdc42, an N-WASP interacting protein. Cdc42 activation is controlled by PKC (protein kinase C). The specificity of Cdc42 targeting to the vesicles, however, is not known. (b) In cultured fibroblasts, PIP5K overexpression or the combination of PDGF (platelet- derived growth factor) and pervanadate (tyrosine phosphatase inhibitor) treatment causes movements of vesicles associated with actin tails. All actin comet tails contain a phosphotyrosine signal, which is essential for Nck recruitment to the vesicles. Nck and WIP are upstream of N-WASP. The specificity of phosphotyrosine signal has not been determined.

assembly provides the driving force for mitochondrial movement. Although there are similarities between the movements of yeast mitochondria, pathogens and endosomes, there are also some fundamental differences. The most prominent one is in the pattern of movement. Arp2/3-complex-mediated movement of Listeria monocytogenes and endosomes has no obvious direction or track dependence. By contrast, yeast mitochondria colocalize with actin cables, bundles of actin filaments that align along the mother–bud axis, and use actin cables as tracks for movement from mother cells to developing daughter cells during cell division [58]. The mechanism underlying the track dependence of mitochondrial movement is not well understood. However, current evidence points towards a mechanism in which proteins including the integral mitochondrial outer membrane proteins Mmm1p and Mdm10p mediate reversible binding of mitochondria to actin cables [59], and actin-binding proteins link newly polymerized http://ticb.trends.com

Targeting of force generators to their cargos Although there has been significant progress in understanding the mechanism of force generation by myosins and the Arp2/3 complex, these force generators are relevant only in the context of their function, which is determined largely by cargo binding. As described above, several signal-transduction mechanisms are required for localization of Arp2/3 complex to endosomes and other vesicles. In addition, the mechanisms of targeting Arp2/3 complex or its activators to pathogen surfaces are well understood [60]. However, the organelle-specific protein(s) that mediate binding of Arp2/3 complex or its activator to endosomal or mitochondrial membranes remains to be determined. By contrast, myosin V binding to organelles can be regulated in two ways. First, mutagenesis studies revealed that two different regions in the carboxyl-terminus tail of myosin V can dictate its binding to secretory vesicles and vacuolar membranes. These sequences and their function are distinct: a mutation in the Myo2p vacuole receptor binding sequence does not disrupt polarized secretion and mutations in the vesicle binding site of Myo2p do not affect vacuole inheritance [31,61]. Second, recent studies support a role for adaptor proteins as direct mediators of myosin binding to organelle membranes. Below, we describe studies leading to identification of myosin V receptors and a new model for receptor-mediated regulation of myosin V activity. A role for Rab GTPase proteins in myosin V targeting comes from localization studies and analysis of cells bearing mutations in or expressing dominant-negative forms of Rab. These studies revealed that: (a) myosin V and specific Rab proteins localize to the same membrane in many cell types; (b) Rab 11p is required for myosin V targeting to secretory vesicles in MDCK cells; (c) Rab8p is required for myosin V binding to endosomal vesicles in HeLa cells; and (d) Rab27a (ashen) is required for targeting of myosin Va to melanosome membranes [61 – 65]. Consistent with this, the Rab yeast homologue Sec4p and the yeast myosin V Myo2p show genetic interactions and are both required for secretory vesicle transport in budding yeast [61,66]. Although two-hybrid evidence indicates that some Rab proteins are capable of direct binding to myosin V [62], at least one Rab protein (Rab27a) might require an adaptor protein to mediate the binding to myosin V. Melanophilin, a rabphilin-like effector protein, is required to recruit myosin Va to melanosome membranes and for actindependent transport of melanosomes in the dendritic periphery of melanocytes. This protein also localizes to melanosome membranes and binds both to Rab27a and to an alternatively spliced exon which encodes a coiled-coil region in the myosin V tail [64,67 – 70]. Taken together, these studies suggest that melanophilin serves as a myosin

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(a)

Melanocyte dendrite membrane

Microtubule-based motor

Melanosome

Microtubule

Melanophilin

Cortical actin filament

Rab27a

MyosinVa

Globular head Coiled-coiled region Globular tail

(b)

Budding yeast

Actin cable

Vacuole

Vac17p

Vac8p

Degraded Vac17p

MyosinV Globular head Coiled-coiled region Globulartail TRENDS in Cell Biology

Fig. 2. Two models for organelle-specific myosin recruitment. (a) Current model for melanosome capture at the peripheral dendritic membrane of melanocytes. Longdistant movement to the cell periphery depends upon the bi-directional movement of melanosomes along microtubules, presumably involving both kinesin and dynein. At the cell periphery, Rab27a-bound melanosomes then recruit melanophilin, a postulated Rab effector, through melanophilin’s N-terminal and in a GTP-dependent fashion. Melanophilin, through its carboxyl-terminal, then binds myosin-Va. This interaction, which requires exon-F of myosin-Va, facilitates cortical capture and short-range movements of the melanosomes within distal, actin-enriched regions of the melanocytic dendrites. (b) Current model for Vac17p-mediate vacuole inheritance. During vegetative growth in budding yeast, Vac17p, in the mother cell, associates to vacuoles via binding to Vac8p, a vacuolar membrane protein. The ‘transport complex’ forms when Vac17p simultaneously binds Myo2p and Vac8p, which can facilitate organelle movement along actin cables towards the bud. In the bud, Vac17p may be targeted for degradation, resulting in the disassembly of the ‘transport complex’ and the deposition or immobilization of vacuoles in the bud.

Va receptor and as an adaptor protein, linking myosin Va to Rab27a (Fig. 2). Meanwhile, work on organelle/particle inheritance in budding yeast has uncovered specific receptors for Myo2p and Myo4p. Recent efforts to uncover a vacuolespecific myosin receptor demonstrated that Vac17p (a) is required for vacuolar inheritance, (b) localizes to vacuoles, and (c) binds directly to a specific region in the Myo2p tail and to Vac8p, an integral vacuolar membrane protein [31,71 – 73]. Thus, like melanophilin, Vac17p might serve as a myosin V receptor and as an adaptor that links myosin V to the vacuolar membrane (Fig. 2). A similar mechanism could underlie targeting of Myo4p to ASH1 mRNA. Recent studies indicate that She3p, a protein that binds to the carboxyl terminus of Myo4p and to the ASH1 mRNA-binding protein She2p, mediates binding of Myo4p to ASH1 mRNA [74,75]. http://ticb.trends.com

Interestingly, Tang et al. [73] showed that VAC17 mRNA and protein levels oscillate in coordination with the cell cycle. Moreover, they found that Vac17p contains a PEST degradation signal and that deletion of the PEST signal resulted in elevated levels of the protein. In light of these findings, Tang et al. propose that PEST signalmediated, bud-specific degradation of the myosin V receptor on vacuoles allows vacuoles to disengage from their force generator in the bud to ensure inheritance. Concluding remarks Although Arp2/3 complex receptors on endogenous vesicles and/or organelles are yet to be identified, recent studies of movement mediated by myosin type V have offered the first lines of evidence for an organelle-specific myosin receptor – a remarkable leap in understanding the mechanism and regulation of organelle movement. Presumably, functional or sequence homologs of Vac8p–Vac17p

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and Rab27a–melanophilin facilitate the specific association of organelles to type-V myosin motor molecules in other organisms. Thus, these studies set the stage for a broad picture of conserved mechanisms of myosin-mediated organelle distribution. Moreover, because the myosin V receptor of the vacuole undergoes cell-cycle-regulated degradation in yeast, it is possible that a similar mechanism is employed to control other membrane–cytoskeleton interactions. The identification of two mechanisms for actin-dependent organelle movement raises one fundamental question: why do some organelles use myosin for actin-based movement, whereas others use the Arp2/3 complex? Although the answer to this question remains to be determined, we suspect that an evolutionary link might exist between Arp2/3 complex localization on endosomes and yeast mitochondria. According to the endosymbiotic hypothesis, mitochondria were derived from prokaryotes taken into the cytoplasm of the early eukaryote by a mechanism similar to that of endocytosis. If this is the case, then the mitochondrial outer membrane might have developed from the endosome membrane. Thus, it is possible that mitochondria retained their force-generating machinery from the endosome when first taken up into the cytosol of the early eukaryote. Moreover, although mitochondria in different cell types use different cytoskeletal networks and force generators for their movement, it is possible that the actin-polymerization-based force generator was maintained and developed into a track-dependent motility mechanism because vegetative yeast contain a highly polarized actin cytoskeleton and because the distances that mitochondria travel in yeast (3 – 6 mm from the mother cell to the bud) are small compared with that of other cells. Ongoing and futures studies might reveal other track-dependent/independent motility events that utilize the Arp2/3 complex for force generation and cargo-specific targeting mechanisms for Arp2/3 complex and/or its activation. Acknowledgements We thank members of the Pon laboratory for critical evaluation of the manuscript. This work was supported by research grants to L.P. from the National Institutes of Health (GM45735) and (GM66037).

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