Stephen J. King

Histone Deacetylase 6 Inhibition Compensates for the Transport Deficit in Huntington's Disease by Increasing Tubulin Acetylation

A defect in microtubule (MT)-based transport contributes to the neuronal toxicity observed in Huntingtons disease (HD). Histone deacetylase (HDAC) inhibitors show neuroprotective effects in this devastating neurodegenerative disorder. We report here that HDAC inhibitors, including trichostatin A (TSA), increase vesicular transport of brain-derived neurotrophic factor (BDNF) by inhibiting HDAC6, thereby increasing acetylation at lysine 40 of α-tubulin. MT acetylation in vitro and in cells causes the recruitment of the molecular motors dynein and kinesin-1 to MTs. In neurons, acetylation at lysine 40 of α-tubulin increases the flux of vesicles and the subsequent release of BDNF. We show that tubulin acetylation is reduced in HD brains and that TSA compensates for the transport- and release-defect phenotypes that are observed in disease. Our findings reveal that HDAC6 inhibition and acetylation at lysine 40 of α-tubulin may be therapeutic targets of interest in disorders such as HD in which intracellular transport is altered.

Cytoplasmic dynein functions as a gear in response to load

Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These ‘nanomachines’ use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dyneins force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load—that is, to shift gears—depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

Multiple-motor based transport and its regulation by Tau

Motor-based intracellular transport and its regulation are crucial to the functioning of a cell. Disruption of transport is linked to Alzheimers and other neurodegenerative diseases. However, many fundamental aspects of transport are poorly understood. An important issue is how cells achieve and regulate efficient long-distance transport. Mounting evidence suggests that many in vivo cargoes are transported along microtubules by more than one motor, but we do not know how multiple motors work together or can be regulated. Here we first show that multiple kinesin motors, working in conjunction, can achieve very long distance transport and apply significantly larger forces without the need of additional factors. We then demonstrate in vitro that the important microtubule-associated protein, tau, regulates the number of engaged kinesin motors per cargo via its local concentration on microtubules. This function of tau provides a previously unappreciated mechanism to regulate transport. By reducing motor reattachment rates, tau affects cargo travel distance, motive force, and cargo dispersal. We also show that different isoforms of tau, at concentrations similar to those in cells, have dramatically different potency. These results provide a well defined mechanism for how altered tau isoform levels could impair transport and thereby lead to neurodegeneration without the need of any other pathway.

Building Complexity: An In Vitro Study of Cytoplasmic Dynein with In Vivo Implications

BACKGROUND Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. RESULTS We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dyneins mechanochemical cycle. CONCLUSIONS Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

This study dissects the recruitment of dynein and dynactin to cargo by a conserved motor adaptor BICD2. It is shown that dynein, dynactin, and BICD2 form a triple complex in vitro and in vivo. Investigation of the properties of this complex by direct visualization of dynein in live cells shows that BICD2-induced dynein transport requires LIS1.

Regulation of Cytoplasmic Dynein ATPase by Lis1

Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport.

Tuning Microtubule-Based Transport Through Filamentous MAPs: The Problem of Dynein

We recently proposed that regulating the single‐to‐multiple motor transition was a likely strategy for regulating kinesin‐based transport in vivo. In this study, we use an in vitro bead assay coupled with an optical trap to investigate how this proposed regulatory mechanism affects dynein‐based transport. We show that tau’s regulation of kinesin function can proceed without interfering with dynein‐based transport. Surprisingly, at extremely high tau levels – where kinesin cannot bind microtubules (MTs) – dynein can still contact MTs. The difference between tau’s effects on kinesin‐ and dynein‐based motility suggests that tau can be used to tune relative amounts of plus‐end and minus‐end‐directed transport. As in the case of kinesin, we find that the 3RS isoform of tau is a more potent inhibitor of dynein binding to MTs. We show that this isoform‐specific effect is not because of steric interference of tau’s projection domains but rather because of tau’s interactions with the motor at the MT surface. Nonetheless, we do observe a modest steric interference effect of tau away from the MT and discuss the potential implications of this for molecular motor structure.

Tuning Multiple Motor Travel Via Single Motor Velocity

Microtubule‐based molecular motors often work in small groups to transport cargos in cells. A key question in understanding transport (and its regulation in vivo) is to identify the sensitivity of multiple‐motor‐based motion to various single molecule properties. Whereas both single‐motor travel distance and microtubule binding rate have been demonstrated to contribute to cargo travel, the role of single‐motor velocity is yet to be explored. Here, we recast a previous theoretical study, and make explicit a potential contribution of velocity to cargo travel. We test this possibility experimentally, and demonstrate a strong negative correlation between single‐motor velocity and cargo travel for transport driven by two motors. Our study thus discovers a previously unappreciated role of single‐motor velocity in regulating multiple‐motor transport.

Casein kinase 2 reverses tail-independent inactivation of kinesin-1

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin.

GSK‐3β Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility

Glycogen synthase kinase 3 (GSK‐3) has been linked to regulation of kinesin‐dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK‐3β in both neurons and non‐neuronal cells. GSK‐3β coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC‐1B and S88/T89 in IC‐2C, have been identified as GSK‐3 targets by both mass spectrometry and site‐directed mutagenesis. These sites are within an Ndel1‐binding domain, and mutation of both sites alters the interaction of ICs with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK‐3β, (ii) an insulin‐sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK‐3β inactivation. Thus, our study connects a well‐characterized insulin‐signaling pathway directly to dynein stimulation via GSK‐3 inhibition.