Margot Lakonishok

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”

Initial Neurite Outgrowth in Drosophila Neurons Is Driven by Kinesin-Powered Microtubule Sliding

Remarkably, forces within a neuron can extend its axon to a target that could be meters away. The two main cytoskeleton components in neurons are microtubules, which are mostly bundled along the axon shaft, and actin filaments, which are highly enriched in a structure at the axon distal tip, the growth cone. Neurite extension has been thought to be driven by a combination of two forces: pushing via microtubule assembly, and/or pulling by an actin-driven mechanism in the growth cone. Here we show that a novel mechanism, sliding of microtubules against each other by the microtubule motor kinesin-1, provides the mechanical forces necessary for initial neurite extension in Drosophila neurons. Neither actin filaments in the growth cone nor tubulin polymerization is required for initial outgrowth. Microtubule sliding in neurons is developmentally regulated and is suppressed during neuronal maturation. As kinesin-1 is highly evolutionarily conserved from Drosophila to humans, it is likely that kinesin-1-powered microtubule sliding plays an important role in neurite extension in many types of neurons across species.

Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape

Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule–microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.

The dynamic properties of intermediate filaments during organelle transport.

Intermediate filament (IF) dynamics during organelle transport and their role in organelle movement were studied using Xenopus laevis melanophores. In these cells, pigment granules (melanosomes) move along microtubules and microfilaments, toward and away from the cell periphery in response to α-melanocyte stimulating hormone (α-MSH) and melatonin, respectively. In this study we show that melanophores possess a complex network of vimentin IFs which interact with melanosomes. IFs form an intricate, honeycomb-like network that form cages surrounding individual and small clusters of melanosomes, both when they are aggregated and dispersed. Purified melanosome preparations contain a substantial amount of vimentin, suggesting that melanosomes bind to IFs. Analyses of individual melanosome movements in cells with disrupted IF networks show increased movement of granules in both anterograde and retrograde directions, further supporting the notion of a melanosome-IF interaction. Live imaging reveals that IFs, in turn, become highly flexible as melanosomes disperse in response to α-MSH. During the height of dispersion there is a marked increase in the rate of fluorescence recovery after photobleaching of GFP-vimentin IFs and an increase in vimentin solubility. These results reveal a dynamic interaction between membrane bound pigment granules and IFs and suggest a role for IFs as modulators of granule movement.

Kinesin-1–powered microtubule sliding initiates axonal regeneration in Drosophila cultured neurons

Microtubule sliding drives initial axon regeneration in Drosophila neurons. Axotomy leads to fast calcium influx and subsequent microtubule reorganization. Kinesin-1 heavy chain drives the sliding of antiparallel microtubules to power axonal regrowth, and the JNK pathway promotes axonal regeneration by enhancing microtubule sliding.

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Significance Generation of mechanical forces by molecular motors is essential for development. Previously, we showed that the microtubule motor kinesin-1 generates forces by sliding microtubules against each other. Here, we show that microtubule sliding by kinesin-1 is important for normal oocyte cytoplasmic rotation, a process required for efficient localization of mRNAs and proteins during oogenesis. Using recently developed imaging technologies (Maple3 photoconversion and SunTag), we discover a previously uncharacterized population of extremely stable microtubules immobilized at the oocyte cortex and demonstrate that free microtubules move against cortically anchored microtubules, generating forces that contribute to cytoplasmic streaming. Because kinesin-1–based sliding is highly conserved from Drosophila to humans, we propose that microtubule sliding is also important for cellular force generation in higher organisms. Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

Ooplasmic flow cooperates with transport and anchorage in Drosophila oocyte posterior determination

The posterior determination of the Drosophila melanogaster embryo is defined by the posterior localization of oskar (osk) mRNA in the oocyte. Defects of its localization result in a lack of germ cells and failure of abdomen specification. A microtubule motor kinesin-1 is essential for osk mRNA posterior localization. Because kinesin-1 is required for two essential functions in the oocyte—transport along microtubules and cytoplasmic streaming—it is unclear how individual kinesin-1 activities contribute to the posterior determination. We examined Staufen, an RNA-binding protein that is colocalized with osk mRNA, as a proxy of posterior determination, and we used mutants that either inhibit kinesin-driven transport along microtubules or cytoplasmic streaming. We demonstrated that late-stage streaming is partially redundant with early-stage transport along microtubules for Staufen posterior localization. Additionally, an actin motor, myosin V, is required for the Staufen anchoring to the actin cortex. We propose a model whereby initial kinesin-driven transport, subsequent kinesin-driven streaming, and myosin V–based cortical retention cooperate in posterior determination.