Biomedical subjects

Fred Chang

Publications and source records attributed to Fred Chang.

At least 19 recordsLinked to original sources

PubMed2006

Shaping the actin cytoskeleton using microtubule tips.

The microtubule cytoskeleton serves as a primary spatial regulator of cell shape. As part of their function, microtubules appear to activate or inhibit the actin cytoskeleton at specific locations at the cell cortex for cell polarization, cell migration and cytokinesis. Recent studies reveal molecular insights into these processes. Regulators of the actin cytoskeleton, such as activators of formin and Rho GTPases, are transported to specific sites on the cortex by riding on the plus ends of microtubules.

Actins↗

PubMed2006

The cell-end factor pom1p inhibits mid1p in specification of the cell division plane in fission yeast.

Intrinsic spatial cues ensure the proper placement of the cell division plane. In the fission yeast Schizosaccharomyces pombe, the position of the nucleus helps to direct the medial positioning of contractile-ring assembly and subsequent cell division . An important factor in this process is mid1p (anillin-like protein), which is a peripheral-membrane protein that forms a broad cortical band of dots overlying the nucleus in interphase and recruits myosin in early mitosis . How mid1p localizes to this cortical band and tracks the nucleus is not clear, especially because its localization is independent of the cytoskeleton . Here, we used a combination of experimental and computational approaches to test mid1p localization mechanisms. We provide evidence that pom1p, a DYRK-family protein kinase that forms a concentration gradient emanating from the nongrowing cell end, inhibits mid1p. In pom1 mutants, mid1p is distributed over half of the cell, covering the nongrowing cell end. This abnormal distribution is established in a dynamic manner in interphase and leads to the formation of misplaced or multiple contractile rings. Our computational and experimental results support a model in which both positive cues from the medial nucleus and negative cues from the cell tips specify the position of the division plane.

Cell Division↗

PubMed2006

Self-organization of microtubule bundles in anucleate fission yeast cells.

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.

Cell Nucleus↗

PubMed2006

Asymmetric microtubule pushing forces in nuclear centering.

Dynamic properties of microtubules contribute to the establishment of spatial order within cells. In the fission yeast Schizosaccharomyces pombe, interphase cytoplasmic microtubules are organized into antiparallel bundles that attach to the nuclear envelope and are needed to position the nucleus at the geometric center of the cell. Here, we show that after the nucleus is displaced by cell centrifugation, these microtubule bundles efficiently push the nucleus back to the center. Asymmetry in microtubule number, length, and dynamics contributes to the generation of force responsible for this unidirectional movement. Notably, microtubules facing the distal cell tip are destabilized when the microtubules in the same bundle are pushing from the proximal cell tip. The CLIP-170-like protein tip1p and the microtubule-bundling protein ase1p are required for this asymmetric regulation of microtubule dynamics, indicating contributions of factors both at microtubule plus ends and within the microtubule bundle. Mutants in these factors are defective in nuclear movement. Thus, cells possess an efficient microtubule-based engine that produces and senses forces for centering the nucleus. These studies may provide insights into mechanisms of asymmetric microtubule behaviors and force sensing in other processes such as chromosome segregation and cell polarization.

Biological Transport↗

PubMed2006

Regulation of cytokinesis by spindle-pole bodies.

In the fission yeast Schizosaccharomyces pombe, cytokinesis is thought to be controlled by the daughter spindle-pole body (SPB) through a regulatory pathway named the septation initiation network (SIN). Here, we demonstrate that laser ablation of both, but not a single SPB, results in failure of cytokinesis. Ablation of only the daughter SPB often leads to activation of the SIN on the mother SPB and successful cytokinesis. Thus, either SPB can drive cytokinesis.

Cell Cycle Proteins↗

PubMed2006

Dynamics of the formin for3p in actin cable assembly.

BACKGROUND: Formins are a conserved family of actin nucleators responsible for the assembly of diverse actin structures such as cytokinetic rings and filopodia. In the fission yeast Schizosaccharomyces pombe, the formin for3p is necessary for the formation of actin cables, which are bundles of short parallel actin filaments that regulate cell polarity. These filaments are largely organized with their barbed ends facing the cell tip, where for3p is thought to function in their assembly. RESULTS: Here, using a functional for3p-3GFP fusion expressed at endogenous levels, we find that for3p localizes to small dots that appear transiently at cell tips and then move away on actin cables at a rate of 0.3 microm/s. These movements were dependent on the continuous assembly of actin in cables, on the ability of for3p to bind actin within its FH2 domain, and on profilin and bud6p, two formin binding proteins that promote formin activity. Bud6p transiently colocalizes with for3p at the cell tip and stays behind at the cell tip when for3p detaches. CONCLUSIONS: These findings suggest a new model for actin cable assembly: a for3p particle is activated and promotes the assembly of a short actin filament at the cell tip for only seconds. For3p and the actin filament may then be released from the cell tip and carried passively into the cell interior by retrograde flow of actin filaments in the cable. These studies reveal a complex and dynamic cycle of formin regulation and actin cable assembly in vivo.

Actin Cytoskeleton↗

PubMed2005

New end take off: regulating cell polarity during the fission yeast cell cycle.

Cell polarization is a major event of the cell cycle and underlies the function of most cells. Cell polarity is often achieved through the coordinated organization of the microtubule and act in cytoskeletons. Dramatic changes in cell polarization occur during the cell cycle and are subject to regulation by cell cycle controls. Cells of the fission yeast Schizosaccharomyces pombe grow by tip extension in a cell cycle-controlled manner. During G2 phase, these cells exhibit a transition in cell polarization known as New End Take Off (NETO), in which monopolar cells initiate bipolar growth. Dynamic microtubules contribute to this process by depositing at cell ends the microtubule plus end proteins tea1p and tea4p, which are necessary for NETO. We discuss here how these proteins may recruit for 3p, a formin responsible for actin nucleation, as well as two other actin binding proteins, bud6p and sla2p, to initiate cell polarization at the new end of the cell. Thus, the study of NETO is revealing a mechanism by which the plus ends of microtubules regulate the spatial organization of actin.

Actins↗

PubMed2005

Role of fission yeast myosin I in organization of sterol-rich membrane domains.

Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of these membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed2005

Dynamic positioning of the fission yeast cell division plane.

A key question in cytokinesis is how the cell division plane is positioned. Whereas microtubules of the mitotic apparatus specify the division site in animal cells, we show here that the nucleus plays this role in the fission yeast Schizosaccharomyces pombe. By centrifuging cells to move the nucleus, we find that the nucleus (or a nuclear-associated structure) actively influences the position of contractile ring assembly during early mitosis. Displacement of the nucleus during this induction period can lead to formation of multiple rings. The nucleus signals its position in a microtubule-independent manner by emitting the protein mid1p. Furthermore, movement of ring fragments together minimizes formation of multiple division sites. These dynamic mechanisms of ring positioning provide a robust coordination of nuclear and cell division.

Cell Nucleus↗

PubMed2005

Effects of {gamma}-tubulin complex proteins on microtubule nucleation and catastrophe in fission yeast.

Although gamma-tubulin complexes (gamma-TuCs) are known as microtubule (MT) nucleators, their function in vivo is still poorly defined. Mto1p (also known as mbo1p or mod20p) is a gamma-TuC-associated protein that recruits gamma-TuCs specifically to cytoplasmic MT organizing centers (MTOCs) and interphase MTs. Here, we investigated gamma-TuC function by analyzing MT behavior in mto1Delta and alp4 (GCP2 homologue) mutants. These cells have free, extra-long interphase MTs that exhibit abnormal behaviors such as cycles of growth and breakage, MT sliding, treadmilling, and hyperstability. The plus ends of interphase and spindle MTs grow continuously, exhibiting catastrophe defects that are dependent on the CLIP170 tip1p. The minus ends of interphase MTs exhibit shrinkage and pauses. As mto1Delta mutants lack cytoplasmic MTOCs, cytoplasmic MTs arise from spindle or other intranuclear MTs that exit the nucleus. Our findings show that mto1p and gamma-TuCs affect multiple properties of MTs including nucleation, nuclear attachment, plus-end catastrophe, and minus-end shrinkage.

Cell Nucleus↗

PubMed2005

Tea4p links microtubule plus ends with the formin for3p in the establishment of cell polarity.

Microtubules regulate actin-based processes such as cell migration and cytokinesis, but molecular mechanisms are not understood. In the fission yeast Schizosaccharomyces pombe, microtubule plus ends regulate cell polarity in part by transporting the kelch repeat protein tea1p to cell ends. Here, we identify tea4p, a SH3 domain protein that binds directly to tea1p. Like tea1p, tea4p localizes to growing microtubule plus ends and to cortical sites at cell ends, and it is necessary for the establishment of bipolar growth. Tea4p binds directly to and recruits the formin for3p, which nucleates actin cable assembly. During "new end take off" (NETO), formation of a protein complex that includes tea1p, tea4p, and for3p is necessary and sufficient for the establishment of cell polarity and localized actin assembly at new cell ends. Our results suggest a molecular mechanism for how microtubule plus ends regulate the spatial distribution of actin assembly.

Actins↗

PubMed2005

Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis.

Plasma membrane subdomains enriched in sphingolipids, cholesterol, and signaling proteins are critical for organization of actin, membrane trafficking, and cell polarity, but the role of such domains in cytokinesis in animal cells is unknown. Here, we show that eggs form a plasma membrane domain enriched in ganglioside G(M1) and cholesterol where tyrosine phosphorylated proteins occur at late anaphase at the contractile ring. The equatorial membrane domain forms by movement-specific lipids and proteins and is dependent on anaphase onset, myosin light chain phosphorylation, actin, and microtubules. Isolated detergent-resistant membranes contain Src and PLCgamma, which become tyrosine phosphorylated at cytokinesis, and whose activation is required for furrow progression. These studies suggest that membrane domains at the cleavage furrow possess a signaling pathway that contributes to cytokinesis.

Actins↗

PubMed2005

Site selection for the cleavage furrow at cytokinesis.

The question of how the site for division of the cytoplasm is determined at the end of mitosis has been studied for over a century, and it remains an active, controversial and fascinating problem in cell biology. This problem draws on the use of several model cell types, with the goal of understanding and identifying how the cell cycle regulates signals between the mitotic apparatus and the cell cortex. Studies in different cell types and using a vast array of techniques reveal different answers: these might reflect differences in experimental approaches, multiple and redundant mechanisms and, importantly, diversity in biology. In this article (which is part of the Cytokinesis series), we present a summary and critique of the major models for the roles of the mitotic apparatus microtubules in stimulating furrow formation at cytokinesis.

Animals↗

PubMed2005

Regulation of actin assembly by microtubules in fission yeast cell polarity.

It has been speculated that microtubule plus ends function to regulate the actin cytoskeleton in processes such as cytokinesis, cell polarization and cell migration. In the fission yeast Schizosaccharomyces pombe, interphase microtubules regulate cell polarity through proteins such as tea1p, a kelch repeat protein, and for3p, a formin that nucleates actin cable assembly at cell tips. Here, we review recent progress on understanding tea1p regulation and function. Microtubules may govern the localization of tea1p by transporting it on the plus ends of microtubules and depositing it directly onto the cell tip when the microtubule catastrophes. The interaction of tea1p with the CLIP170 protein tip1p is responsible for its localization at growing microtubule plus ends. Tea1p may regulate cell polarity by associating with large 'polarisome' complexes that include for3p. For3p is present at both cell tips, but is not on the microtubules. Tea1p is needed to localize the formin to establish polarized cell growth at cell tips that have not grown previously. These studies begin to elucidate a molecular pathway for how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.

Actins↗

PubMed2004

Organization of a sterol-rich membrane domain by cdc15p during cytokinesis in fission yeast.

Many membrane processes occur in discrete membrane domains containing lipid rafts, but little is known about how these domains are organized and positioned. In the fission yeast Schizosaccharomyces pombe, a sterol-rich membrane domain forms at the cell-division site. Here, we show that formation of this membrane domain is independent of the contractile actin ring, septation, mid1p and the septins, and also requires cdc15p, an essential contractile ring protein that associates with lipid rafts. cdc15 mutants have membrane domains in the shape of spirals. Overexpression of cdc15p in interphase cells induces abnormal membrane domain formation in an actin-independent manner. We propose that cdc15p functions to organize lipid rafts at the cleavage site for cytokinesis.

Cell Cycle↗

PubMed2004

Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B.

INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe. RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles. CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast.

Anaphase↗

PubMed2004

Regulation of a formin complex by the microtubule plus end protein tea1p.

The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Deltabud6Deltatea1Delta triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.

Actin Cytoskeleton↗

PubMed2004

HIV-1 Vpr induces defects in mitosis, cytokinesis, nuclear structure, and centrosomes.

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 15-kDa accessory protein that contributes to several steps in the viral replication cycle and promotes virus-associated pathology. Previous studies demonstrated that Vpr inhibits G2/M cell cycle progression in both human cells and in the fission yeast Schizosaccharomyces pombe. Here, we report that, upon induction of vpr expression, fission yeast exhibited numerous defects in the assembly and function of the mitotic spindle. In particular, two spindle pole body proteins, sad1p and the polo kinase plo1p, were delocalized in vpr-expressing yeast cells, suggesting that spindle pole body integrity was perturbed. In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted. Similar Vpr-induced defects in mitosis and cytokinesis were observed in human cells, including aberrant mitotic spindles, multiple centrosomes, and multinucleate cells. These defects in cell division and centrosomes might account for some of the pathological effects associated with HIV-1 infection.

Cell Cycle↗