Stephen L. Rogers

Molecular requirements for actin-based lamella formation in Drosophila S2 cells

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of ∼90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the ‘Pac-Man’ model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by ‘chewing up’ microtubule tracks. In the ‘poleward flux’ model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

Myosin cooperates with microtubule motors during organelle transport in melanophores

Melanophores offer an outstanding system for the study of intracellular motility. These cells aggregate their pigment-filled melanosomes to the cell center or disperse them throughout the cytoplasm in response to hormonal modulation of intracellular cyclic AMP levels in order to effect color changes in lower vertebrates [1]. Previous work from our laboratory demonstrated a role for microtubule-based motors in melanosome transport and we succeeded in reconstituting their regulated motility along microtubules in vitro [2,3]. Here we demonstrate that, in addition to microtubule-mediated motility, melanosomes purified from Xenopus melanophores exhibit unidirectional movement along actin filaments in vitro as well. Immunoblotting analysis shows that these organelles possess the actin-based organelle motor, myosin-V. In vivo, melanosomes are able to slowly disperse in the absence of microtubules, and this slow dispersion requires the integrity of the actin cytoskeleton. Furthermore, in cells with dispersed pigment, disruption of filamentous actin induces a rapid, microtubule-dependent aggregation of melanosomes to the cell center. Our results, together with the accompanying paper by Rodionov et al. [4], demonstrate that the concerted efforts of both microtubule-based and actin-based motors are required for proper melanosome distribution in melanophores. This is the first example of a biochemically defined organelle in possession of both plus-end and minus-end directed microtubule motors and a myosin; coordinated activity of all three motors is essential for organelle motility in vivo.

Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner.

Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs) , have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Galpha subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation and morphogenesis . Using Drosophila S2 cells as a model system, we show that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, we found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. We propose that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Galpha activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development.

The SCF Slimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication.

Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCFSlimb as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCFSlimb target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

Membrane trafficking, organelle transport, and the cytoskeleton

Cytoskeleton-associated motor proteins typically drive organelle movements in eukaryotic cells in a manner that is tightly regulated, both spatially and temporally. In the past year, a novel organelle transport mechanism utilizing actin polymerization was described. Important advances were also made in the assignment of functions to several new motors and in our understanding of how motor proteins are regulated during organelle transport. In addition, insights were gained into how and why organelles are transported cooperatively along the microtubule and actin cytoskeletons, and into the importance of motor-mediated transport in the organization of the cytoskeleton itself.

Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end

EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1s COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.

Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.

Culture of Drosophila S2 cells and their use for RNAi-mediated loss-of-function studies and immunofluorescence microscopy

Cultured Drosophila cell lines have become an increasingly popular model system for cell biological and functional genomic studies. One of the most commonly used lines, S2 cells, is particularly useful as it is easy to grow and maintain in the lab, is highly susceptible to gene inhibition using RNAi and is well suited to high-resolution light microscopic assays. Here, we provide protocols for the routine culture and RNAi treatment of S2 cells and methods to prepare these cells for fluorescence microscopy. Using these techniques, loss-of-function experiments may be performed after 4–7 d of RNAi-mediated protein depletion.

Microtubule binding by dynactin is required for microtubule organization but not cargo transport

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150glued, binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150glued, we replaced the wild-type p150glued in Drosophila melanogaster S2 cells with mutant ΔN-p150 lacking residues 1–200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150glued with ΔN-p150glued has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150glued has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.