Rab32 Regulates Melanosome Transport in Xenopus Melanophores by Protein Kinase A Recruitment

SummaryIntracellular transport is essential for cytoplasm organization, but mechanisms regulating transport are mostly unknown. In Xenopus melanophores, melanosome transport is regulated by cAMP-dependent protein kinase A (PKA) [1]. Melanosome aggregation is triggered by melatonin, whereas dispersion is induced by melanocyte-stimulating hormone (MSH) [2]. The action of hormones is mediated by cAMP: High cAMP in MSH-treated cells stimulates PKA, whereas low cAMP in melatonin-treated cells inhibits it. PKA activity is typically restricted to specific cell compartments by A-kinase anchoring proteins (AKAPs) [3]. Recently, Rab32 has been implicated in protein trafficking to melanosomes [4] and shown to function as an AKAP on mitochondria [5]. Here, we tested the hypothesis that Rab32 is involved in regulation of melanosome transport by PKA. We demonstrated that Rab32 is localized to the surface of melanosomes in a GTP-dependent manner and binds to the regulatory subunit RIIα of PKA. Both RIIα and Cβ subunits of PKA are required for transport regulation and are recruited to melanosomes by Rab32. Overexpression of wild-type Rab32, but not mutants unable to bind PKA or melanosomes, inhibits melanosome aggregation by melatonin. Therefore, in melanophores, Rab32 is a melanosome-specific AKAP that is essential for regulation of melanosome transport

Interactions and regulation of molecular motors in Xenopus melanophores

Many cellular components are transported using a combination of the actin- and microtubule-based transport systems. However, how these two systems work together to allow well-regulated transport is not clearly understood. We investigate this question in the Xenopus melanophore model system, where three motors, kinesin II, cytoplasmic dynein, and myosin V, drive aggregation or dispersion of pigment organelles called melanosomes. During dispersion, myosin V functions as a “molecular ratchet” to increase outward transport by selectively terminating dynein-driven minus end runs. We show that there is a continual tug-of-war between the actin and microtubule transport systems, but the microtubule motors kinesin II and dynein are likely coordinated. Finally, we find that the transition from dispersion to aggregation increases dynein-mediated motion, decreases myosin V–mediated motion, and does not change kinesin II–dependent motion. Down-regulation of myosin V contributes to aggregation by impairing its ability to effectively compete with movement along microtubules

Dynactin is required for bidirectional organelle transport

Kinesin II is a heterotrimeric plus end–directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II–mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II–associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530–793 of XKAP and aa 600–811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities

Chemical structure-guided design of dynapyrazoles, potent cell-permeable dynein inhibitors with a unique mode of action

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition

Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors.

Xenopus melanophores have pigment organelles or melanosomes which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. Melanosomes are transported by microtubule motors, kinesin-2 and cytoplasmic dynein, and an actin motor, myosin-V. We explored the regulation of melanosome transport along microtubules in vivo by using a new fast-tracking routine, which determines the melanosome position every 10 ms with 2-nm precision. The velocity distribution of melanosomes transported by cytoplasmic dynein or kinesin-2 under conditions of aggregation and dispersion presented several peaks and could not be fit with a single Gaussian function. We postulated that the melanosome velocity depends linearly on the number of active motors. According to this model, one to three dynein molecules transport each melanosome in the minus-end direction. The transport in the plus-end direction is mainly driven by one to two copies of kinesin-2. The number of dyneins transporting a melanosome increases during aggregation, whereas the number of active kinesin-2 stays the same during aggregation and dispersion. Thus, the number of active dynein molecules regulates the net direction of melanosome transport. The model also shows that multiple motors of the same polarity cooperate during the melanosome transport, whereas motors of opposite polarity do not compete

Melanosomes transported by myosin-V in Xenopus melanophores perform slow 35 nm steps.

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20-80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes' motion

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection.

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed