Groundbreaking research published by BIO5 scientists and their collaborators

 

PubMed Articles

Search form

Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). We report that Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of gamma-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing gamma-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden our understanding of the molecular machinery used to move chromosomes.

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150(glued), binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150(glued), we replaced the wild-type p150(glued) in Drosophila melanogaster S2 cells with mutant DeltaN-p150 lacking residues 1-200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150(glued) with DeltaN-p150(glued) has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150(glued) has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.

Accurate and timely chromosome segregation is a task performed within meiotic and mitotic cells by a specialized force-generating structure--the spindle. This micromachine is constructed from numerous proteins, most notably the filamentous microtubules that form a structural framework for the spindle and also transmit forces through it. Poleward flux is an evolutionarily conserved mechanism used by spindle microtubules both to move chromosomes and to regulate spindle length. Recent studies have identified a microtubule-depolymerizing kinesin as a key force-generating component required for flux. On the basis of these findings, we propose a new model for flux powered by a microtubule-disassembly mechanism positioned at the spindle pole. In addition, we use the flux model to explain the results of spindle manipulation experiments to illustrate the importance of flux for proper chromosome positioning.

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here, we report that two proteins of the Drosophila melanogaster kinesin-13 family, KLP10A and KLP59C, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; KLP10A stimulates catastrophe (a switch from growth to shrinkage) whereas KLP59C suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live analyses of cells expressing tagged kinesins reveal that KLP10A and KLP59C target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that KLP10A is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the kinesin-13 family divide the labour of microtubule depolymerization.

Anaphase A chromatid-to-pole motion is fundamental for proper chromosome segregation in most systems. During the past several decades, two models for the mechanism of anaphase A have come to prominence. The Pacman model posits that chromatids induce the depolymerization of microtubule plus-ends embedded in kinetochores, thereby 'chewing' their way poleward. Alternatively, the Poleward-flux model posits that chromatids are 'reeled-in' to poles by the continual depolymerization of the minus-ends of kinetochore-associated microtubules, which are focused at spindle poles. In a recent study, we reported that anaphase A in Drosophila requires the depolymerization of both ends of kinetochore-associated microtubules, simultaneously. This is driven by two members of the Kin I subfamily of kinesins, termed KLP59C and KLP10A, which target specifically to chromatids and spindle poles, respectively. We have termed this hybrid of Pacman and Poleward flux the Kin I-dependent Pacman-flux mechanism for anaphase A. Here, we discuss the implications of these findings and explore potential additional components required to drive chromatid-to-pole motion by such a mechanism.

During anaphase identical sister chromatids separate and move towards opposite poles of the mitotic spindle. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at the spindle pole. Two models have been proposed to account for the movement of chromatids during anaphase. In the 'Pac-Man' model, kinetochores induce the depolymerization of kinetochore microtubules at their plus ends, which allows chromatids to move towards the pole by 'chewing up' microtubule tracks. In the 'poleward flux' model, kinetochores anchor kinetochore microtubules and chromatids are pulled towards the poles through the depolymerization of kinetochore microtubules at the minus ends. Here, we show that two functionally distinct microtubule-destabilizing KinI kinesin enzymes (so named because they possess a kinesin-like ATPase domain positioned internally within the polypeptide) are responsible for normal chromatid-to-pole motion in Drosophila. One of them, KLP59C, is required to depolymerize kinetochore microtubules at their kinetochore-associated plus ends, thereby contributing to chromatid motility through a Pac-Man-based mechanism. The other, KLP10A, is required to depolymerize microtubules at their pole-associated minus ends, thereby moving chromatids by means of poleward flux.

Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.

The mechanical events of mitosis depend on the action of microtubules and mitotic motors, but whether these spindle components act alone or in concert with a spindle matrix is an important question.

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.

To improve our understanding of the roles of microtubule cross-linking motors in mitosis, we analyzed two sea urchin embryonic kinesin-related proteins. It is striking to note that both of these proteins behave as homotetramers, but one behaves as a more compact molecule than the other. These observations suggest that these two presumptive motors could cross-link microtubules into bundles with different spacing. Both motors localize to mitotic spindles, and antibody microinjection experiments suggest that they have mitotic functions. Thus, one of these kinesin-related proteins may cross-link spindle microtubules into loose bundles that are "tightened" by the other.

The mitotic spindle uses microtubule-based motor proteins to assemble itself and to segregate sister chromatids. It is becoming clear that motors invoke several distinct mechanisms to generate the forces that drive mitosis. Moreover, in carrying out its function, the spindle appears to pass through a series of transient steady-state structures, each established by a delicate balance of forces generated by multiple complementary and antagonistic motors. Transitions from one steady state to the next can occur when a change in the activity of a subset of mitotic motors tips the balance.

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.

Eukaryotic cells must build a complex infrastructure of microtubules (MTs) and associated proteins to carry out a variety of functions. A growing body of evidence indicates that a major function of MT-associated motor proteins is to assemble and maintain this infrastructure. In this context, we examine the mechanisms utilized by motors to construct the arrays of MTs and associated proteins contained within the mitotic spindle, neuronal processes, and ciliary axonemes. We focus on the capacity of motors to drive the 'sliding filament mechanism' that is involved in the construction and maintenance of spindles, axons and dendrites, and on a type of particle transport called 'intraflagellar transport' which contributes to the assembly and maintenance of axonemes.

It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear. Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is down-regulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B.

Several novel members of the kinesin superfamily, until now identified only in plants, are unique in their ability to bind calmodulin in the presence of Ca(2+). Here, we identify the first such kinesin in an animal system. Sequence analysis of this new motor, called kinesin-C, predicts that it is a large carboxy-terminal kinesin, 1624 amino acid residues in length, with a predicted molecular mass of 181 kDa. Kinesin-C is predicted to contain a kinesin motor domain at its carboxy terminus, linked to a segment of alpha-helical coiled-coil 950 amino acid residues long, ending with an amino-terminal proline-rich tail domain. A putative calmodulin-binding domain resides at the extreme carboxy terminus of the motor polypeptide, and recombinant kinesin-C binds to a calmodulin-affinity column in a Ca(2+)-dependent fashion. The presence of this novel calmodulin-binding motor in sea urchin embryos suggests that it plays a critical role in Ca(2+)-dependent events during early sea urchin development.

No abstract given.

A novel approach to specifically target tumor cells for detection and treatment is the proposed use of heteromultivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligands with two or more different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the human melanocortin 4 receptor (MC4R) expressed in combination with either the human delta-opioid receptor (deltaOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptors were characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-alpha-MSH, and Eu-CCK8 for the deltaOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a deltaOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, compared with cells with single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers.

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.

Colocalization, in which images of two or more fluorescent markers are overlaid, and coincidence between the probes is measured or displayed, is a common analytical tool in cell biology. Interpreting the images and the meaning of this identified coincidence is difficult in the absence of basic information about the acquisition parameters. In this commentary, we highlight important factors in the acquisition of images used to demonstrate colocalization, and we discuss the minimum information that authors should include in a manuscript so that a reader can interpret both the fluorescent images and any observed colocalization.

Pages