Richard B. Vallee

A role for the lissencephaly gene LIS1 in mitosis and cytoplasmic dynein function

Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.

LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

Dual subcellular roles for LIS1 and dynein in radial neuronal migration in live brain tissue.

During brain development, neural precursor cells migrate along radial glial fibers to populate the neocortex. RNA interference (RNAi) of the lissencephaly gene LIS1 (also known as PAFAH1b1) inhibits somal movement but not process extension of neural precursors in live brain slices. Here we report imaging of the subcellular events accompanying neural precursor migration and the effects of LIS1, cytoplasmic dynein and myosin II inhibition. Centrosomes move continuously and often far in advance of nuclei, which show extreme saltatory behavior. LIS1 and dynein RNAi inhibit centrosomal and nuclear movement independently, whereas myosin II inhibition blocks only nuclear translocation. Imaging of the microtubule end-binding protein 3 (EB3) reveals a centrosome-centered array of microtubules in live neural precursors under all conditions examined. Dynein is concentrated both at a swelling in the leading process reported to initiate each migratory cycle and in the soma. Thus, dynein pulls on the microtubule network from the swelling. The nucleus is transported along the trailing microtubules by dynein assisted by myosin II.

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.

Asymmetric centrosome inheritance maintains neural progenitors in the neocortex

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.

An extended microtubule-binding structure within the dynein motor domain

Flagellar dynein was discovered over 30 years ago as the first motor protein capable of generating force along microtubules. A cytoplasmic form of dynein has also been identified which is involved in mitosis and a wide range of other intracellular movements (reviewed in ref. 3). Rapid progress has been made on understanding the mechanism of force production by kinesins and myosins. In contrast, progress in understanding the dyneins has been limited by their great size (relative molecular mass 1,000K–2,000K) and subunit complexity. We now report evidence that the entire carboxy-terminal two-thirds of the 532K force-producing heavy chain subunit is required for ATP-binding activity. We further identify a microtubule-binding domain, which, surprisingly, lies well downstream of the entire ATPase region and is predicted to form a hairpin-like stalk. Direct ultrastructural analysis of a recombinant fragment confirms this model, and suggests that the mechanism for dynein force production differs substantially from that of other motor proteins.

Targeting of Motor Proteins

Microtubules are responsible for chromosome segregation and the movement and reorganization of membranous organelles. Many aspects of microtubule-based motility can be attributed to the action of motor proteins, producing force directed toward either end of microtubules. How these proteins are targeted to the appropriate organellar sites within the cell, however, has remained a mystery. Recent work has begun to define the targeting mechanism for two well-studied motor proteins, kinesin and cytoplasmic dynein.

LIS1 and NudE Induce a Persistent Dynein Force-Producing State

Cytoplasmic dynein is responsible for many aspects of cellular and subcellular movement. LIS1, NudE, and NudEL are dynein interactors initially implicated in brain developmental disease but now known to be required in cell migration, nuclear, centrosomal, and microtubule transport, mitosis, and growth cone motility. Identification of a specific role for these proteins in cytoplasmic dynein motor regulation has remained elusive. We find that NudE stably recruits LIS1 to the dynein holoenzyme molecule, where LIS1 interacts with the motor domain during the prepowerstroke state of the dynein crossbridge cycle. NudE abrogates dynein force production, whereas LIS1 alone or with NudE induces a persistent-force dynein state that improves ensemble function of multiple dyneins for transport under high-load conditions. These results likely explain the requirement for LIS1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton as well as the particular sensitivity of migrating neurons to reduced LIS1 expression.

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170–dynein interactions and in coordinating dynein cargo-binding and motor activities.

Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein

n higher eukaryotes, secretory and plasma-membrane proteins are transported from the endoplasmic reticulum (ER) to a central Golgi complex and subsequently packaged into membrane-bound carriers for delivery to the cell surface. The long-distance transport of post-Golgi organelles to the tips of axons or to developing hyphal extensions in Neurospora crassa shows an absolute requirement for microtubules and microtubule-associated motors. In contrast, microtubule disruption only moderately attenuates Golgi-toplasma-membrane transport in fibroblasts and randomizes surface delivery of select proteins in epithelial cells. The observed preservation of biosynthetic transport after microtubule disruption is probably due to the extensive fragmentation and redistribution of Golgi mini-stacks to regions immediately adjacent to both the ER and the plasma membrane. Here we have designed experiments to test the hypothesis that when the characteristic central localization of the Golgi is preserved, microtubules, kinesin and the GTPase dynamin are essential for post-Golgi trafficking. We ruled out a pharmacological approach to tackling this problem because we found that microtubule antagonists caused dispersal of the Golgi complex before complete microtubule disassembly occurred (see Supplementary Information). Instead, we microinjected functionblocking anti-kinesin antibodies HD and SUK-4 or cDNAs encoding a dominant-negative form of dynamin into cells expressing a green fluorescent protein (GFP)-tagged apical-membrane protein, p75. We show that kinesin and dynamin are required for different stages of post-Golgi transport. During a 2.5-h transport block at 20 °C, newly synthesized p75– GFP translocated from the ER to a juxtanuclear region (Fig. 1a, control, 0 min) and co-localized with Golgi/trans-Golgi network (TGN) markers (Fig. 1b). Within 4 h after shifting to the permissive temperature for transport, 32 °C, 81% of p75–GFP translocated from the Golgi to the plasma membrane (Fig. 1a, control, 240 min). The emptying rate of p75–GFP from the Golgi correlated with its arrival at the cell surface, as determined by immunocytochemical analysis of p75 in injected cells (data not shown) and by pulse– chase, surface-biotinylation assays of p75 or p75–GFP in stable MDCK transfectants (see Supplementary Information). Normal trafficking of p75 was unaffected by the GFP tag: microinjected p75–GFP was selectively delivered to the apical membrane of confluent, polarized MDCK cells (data not shown). I