In JoVE (1)

Other Publications (22)

Articles by Jeffrey K. Moore in JoVE

Other articles by Jeffrey K. Moore on PubMed

Retinal Detachment in Eyes Undergoing Pars Plana Vitrectomy for Removal of Retained Lens Fragments

Ophthalmology. Apr, 2003  |  Pubmed ID: 12689890

To investigate the incidence and outcomes of retinal detachment (RD) associated with retained lens fragments removed by pars plana vitrectomy (PPV).

Clinical Features and Outcomes of Pars Plana Vitrectomy in Patients with Retained Lens Fragments

Ophthalmology. Aug, 2003  |  Pubmed ID: 12917174

To investigate the clinical features, visual acuity outcomes, and adverse events in patients with retained lens fragments managed by pars plana vitrectomy (PPV).

Immediate Postoperative Use of a Topical Agent to Prevent Intraocular Pressure Elevation After Pars Plana Vitrectomy with Gas Tamponade

Archives of Ophthalmology (Chicago, Ill. : 1960). May, 2004  |  Pubmed ID: 15136318

To determine whether a single topical aqueous suppressant applied immediately after pars plana vitrectomy with long-acting gas tamponade prevents intraocular pressure (IOP) elevation.

The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9p

Molecular Biology of the Cell. Jan, 2006  |  Pubmed ID: 16236795

Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1delta mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5delta mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.

The CLIP-170 Orthologue Bik1p and Positioning the Mitotic Spindle in Yeast

Current Topics in Developmental Biology. 2006  |  Pubmed ID: 17118263

Bik1p is the yeast Saccharomyces cerevisiae representative of the CLIP-170 family of microtubule plus-end tracking proteins. Bik1p shares a number of similarities with its mammalian counterpart CLIP-170, including an important role in dynein function. However, Bik1p and CLIP-170 differ in several significant ways, including the mechanisms utilized to track microtubule plus ends. In addition to presenting functional comparisons between Bik1p and CLIP-170, we provide sequence analyses that reveal previously unrecognized similarities between Bik1p and its animal counterparts. We examine in detail what is known about the functions of Bik1p and consider the various roles that Bik1p plays in positioning the yeast mitotic spindle. This chapter also highlights several recent findings, including the contribution of Bik1p to the yeast mating pathway.

The Cyclin-dependent Kinase Cdc28p Regulates Multiple Aspects of Kar9p Function in Yeast

Molecular Biology of the Cell. Apr, 2007  |  Pubmed ID: 17251549

During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.

Retinal Breaks Observed During Pars Plana Vitrectomy

American Journal of Ophthalmology. Jul, 2007  |  Pubmed ID: 17509513

To quantitate the frequency and features of retinal breaks discovered at the time of vitrectomy and to evaluate the outcomes with prophylactic treatment.

Vehicle-controlled, Double-blind, Randomized Study of Imiquimod 5% Cream Applied 3 Days Per Week in One or Two Courses of Treatment for Actinic Keratoses on the Head

Journal of the American Academy of Dermatology. Aug, 2007  |  Pubmed ID: 17512087

A shorter dosing regimen of imiquimod for the treatment of actinic keratosis may be effective, with long-term clinical benefits.

Dynactin Function in Mitotic Spindle Positioning

Traffic (Copenhagen, Denmark). Apr, 2008  |  Pubmed ID: 18221362

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150(Glued), dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150(Glued) was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast.

Neurodegeneration Mutations in Dynactin Impair Dynein-dependent Nuclear Migration

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2009  |  Pubmed ID: 19279216

Neurodegenerative disease in humans and mice can be caused by mutations affecting the microtubule motor dynein or its biochemical regulator, dynactin, a multiprotein complex required for dynein function (1-4). A single amino acid change, G59S, in the conserved cytoskeletal-associated protein glycine-rich (CAP-Gly) domain of the p150(glued) subunit of dynactin can cause motor neuron degeneration in humans and mice, which resembles ALS (2, 5-8). The molecular mechanism by which G59S impairs the function of dynein is not understood. Also, the relevance of the CAP-Gly domain for dynein motility has not been demonstrated in vivo. Here, we generate a mutant that is analogous to G59S in budding yeast, and show that this mutation produces a highly specific phenotype related to dynein function. The effect of the point mutation is identical to that of complete loss of the CAP-Gly domain. Our results demonstrate that the CAP-Gly domain has a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus, but it is otherwise dispensable for dynein-based movement. The need for this function appears to be context-dependent, and we speculate that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement.

Function of Dynein in Budding Yeast: Mitotic Spindle Positioning in a Polarized Cell

Cell Motility and the Cytoskeleton. Aug, 2009  |  Pubmed ID: 19402153

Cytoplasmic dynein is a microtubule motor that powers minus-end-directed motility in a variety of biological settings. The budding yeast, Saccharomyces cerevisiae, has been a useful system for the study of dynein, due to its molecular genetics and cell biology capabilities, coupled with the conservation of dynein-pathway proteins. In this review we discuss how budding yeast use dynein to manipulate the position of the mitotic spindle and the nucleus during cell division, using cytoplasmic microtubules, and we describe our current understanding of the genes required for dynein function. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

The Spindle Position Checkpoint Requires Positional Feedback from Cytoplasmic Microtubules

Current Biology : CB. Dec, 2009  |  Pubmed ID: 19913426

The objective of mitosis is to provide a copy of the genome to each progeny of a cell division. This requires the separation of duplicate chromatids by the spindle apparatus and the delivery of one set of chromosomes to each of the daughter cells. In budding yeast, the fidelity of chromosome delivery depends on the spindle position checkpoint, which prolongs mitosis until one end of the anaphase spindle arrives in the bud. Here we tested the hypothesis that the activity of the spindle position checkpoint depends on persistent interactions between cytoplasmic microtubules and the mother-bud neck, the future site of cytokinesis. We used laser ablation to disrupt microtubule interactions with the bud neck, and we found that loss of microtubules from the neck leads to mitotic exit in a majority of checkpoint-activated cells. Our findings suggest that cytoplasmic microtubules are used to monitor the location of the spindle in the dividing cell and, in the event of positioning errors, relay a signal to inhibit mitotic exit until the spindle is appropriately positioned.

Coordinating Mitosis with Cell Polarity: Molecular Motors at the Cell Cortex

Seminars in Cell & Developmental Biology. May, 2010  |  Pubmed ID: 20109571

In many cell divisions, the position of the spindle apparatus is coordinated with polarity signals at the cell cortex so that copies of the genome are delivered to regions of the cell that are designated for differential inheritance by the two progeny. To coordinate spindle position with cell polarity, the spindle interfaces with elements on the cortex, where molecular motors often produce the forces that power displacement. Here we describe the molecular pathways by which cortical motors translocate the spindle in budding yeast, where the mechanisms are understood relatively well, and we compare these pathways to spindle positioning processes in metazoan systems, where the molecular details are less well understood.

The Spindle Position Checkpoint is Coordinated by the Elm1 Kinase

The Journal of Cell Biology. Nov, 2010  |  Pubmed ID: 21041444

How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother-daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization.

Functional Interaction Between Dynein Light Chain and Intermediate Chain is Required for Mitotic Spindle Positioning

Molecular Biology of the Cell. Aug, 2011  |  Pubmed ID: 21633107

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.

Dynein and Dynactin Leverage Their Bivalent Character to Form a High-affinity Interaction

PloS One. 2013  |  Pubmed ID: 23577064

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

Stopped in Its Tracks: Negative Regulation of the Dynein Motor by the Yeast Protein She1

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Aug, 2013  |  Pubmed ID: 23666903

How do cells direct the microtubule motor protein dynein to move cellular components to the right place at the right time? Recent studies in budding yeast shed light on a new mechanism for directing dynein, involving the protein She1. She1 restricts where and when dynein moves the nucleus and mitotic spindle. Experiments with purified proteins show that She1 binds to microtubules and inhibits dynein by stalling the motor on its track. Here I describe what we have learned so far about She1, based on a combination of genetic, cell biology, and biophysical approaches. These findings set the stage for further interrogation of the She1 mechanism, and raise the question of whether similar mechanisms exist in other species.

Genome-wide Analysis Reveals Novel and Discrete Functions for Tubulin Carboxy-terminal Tails

Current Biology : CB. Jun, 2014  |  Pubmed ID: 24835459

Microtubules (MTs) support diverse transport and force generation processes in cells. Both α- and β-tubulin proteins possess carboxy-terminal tail regions (CTTs) that are negatively charged, intrinsically disordered, and project from the MT surface where they interact with motors and other proteins. Although CTTs are presumed to play important roles in MT networks, these roles have not been determined in vivo.

Tubulin Cofactors and Arl2 Are Cage-like Chaperones That Regulate the Soluble αβ-tubulin Pool for Microtubule Dynamics

ELife. Jul, 2015  |  Pubmed ID: 26208336

Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.

Novel α-tubulin Mutation Disrupts Neural Development and Tubulin Proteostasis

Developmental Biology. Jan, 2016  |  Pubmed ID: 26658218

Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.

The Negatively Charged Carboxy-terminal Tail of β-tubulin Promotes Proper Chromosome Segregation

Molecular Biology of the Cell. Jun, 2016  |  Pubmed ID: 27053662

Despite the broadly conserved role of microtubules in chromosome segregation, we have a limited understanding of how molecular features of tubulin proteins contribute to the underlying mechanisms. Here we investigate the negatively charged carboxy-terminal tail domains (CTTs) of α- and β-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating β-CTT causes elevated rates of chromosome loss and cell cycle delay. Complementary live-cell imaging and electron tomography show that β-CTT is necessary to properly position kinetochores and organize microtubules within the assembling spindle. We identify a minimal region of negatively charged amino acids that is necessary and sufficient for proper chromosome segregation and provide evidence that this function may be conserved across species. Our results provide the first in vivo evidence of a specific role for tubulin CTTs in chromosome segregation. We propose that β-CTT promotes the ordered segregation of chromosomes by stabilizing the spindle and contributing to forces that move chromosomes toward the spindle poles.

Cingulin and Actin Mediate Midbody-dependent Apical Lumen Formation During Polarization of Epithelial Cells

Nature Communications. Aug, 2016  |  Pubmed ID: 27484926

Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.

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