Phospholipase D signaling regulates microtubule organization in the fucoid alga Silvetia compressa

Recent studies in higher plants or animals have shown that phospholipase D (PLD) signaling regulates many aspects of development, including organization of microtubules (MTs), actin and the endomembrane system. PLD hydrolyzes structural phospholipids to form the second messenger phosphatidic acid (PA). To begin to understand the signaling pathways and molecules that regulate cytoskeletal and endomembrane arrays during early development in the brown alga, Silvetia compressa, we altered PLD activity by applying butyl alcohols to zygotes. 1-Butanol activates PLD and is a preferred substrate, primarily forming phosphatidyl butanol (P-butanol), which is not a signaling molecule. Treatment with 1-butanol inhibited cell division and cytokinesis but not photopolarization or germination, suggesting an MT-based effect. Immunolabeling revealed that 1-butanol treatment rapidly disrupted MT arrays and caused zygotes to arrest in metaphase. MT arrays recovered rapidly following butanol washout, but subsequent development depended on the timing of the treatment regime. Additionally, treatment with 1-butanol early in development disrupted endomembrane organization, known to require functional MTs. Interestingly, treatment with higher concentrations of 2-butanol, which also activates PLD, mimicked the effects of 1-butanol. In contrast, the control t-butanol had no effect on MTs or development. These results indicate that S. compressa zygotes utilize PLD signaling to regulate MT arrays. In contrast, PLD signaling does not appear to regulate actin arrays or endomembrane trafficking directly. This is the first report describing the signaling pathways that regulate cytoskeletal organization in the stramenopile (heterokont) lineage.     

Kinesin-13s in mitosis: Key players in the spatial and temporal organization of spindle microtubules

Dynamic microtubules are essential for the process of mitosis. Thus, elucidating when, where, and how microtubule dynamics are regulated is key to understanding this process. One important class of proteins that directly regulates microtubule dynamics is the Kinesin-13 family. Kinesin-13 proteins induce depolymerization uniquely from both ends of the microtubule. This activity coincides with their cellular localization and with their ability to regulate microtubule dynamics to control spindle assembly and kinetochore-microtubule attachments. In this review, we highlight recent findings that dissect the important actions of Kinesin-13 family members and summarize important studies on the regulation of their activity by phosphorylation and by protein–protein interactions.     

Cortical actin filaments form rapidly during photopolarization and are required for the development of calcium gradients in Pelvetia compressa zygotes

Previous research has shown that cortical gradients of cytosolic Ca(2+) are formed during the photopolarization of Pelvetia compressa zygotes, with elevated Ca(2+) on the shaded hemisphere that will become the site of rhizoid germination. We report here that the marine sponge toxin, latrunculin B, which blocks photopolarization at nanomolar concentrations, inhibited the formation of the light-driven Ca(2+) gradients. Using low concentrations of microinjected fluorescent phalloidin as a tracer for actin filaments, we found that exposure to light induced a striking increase in actin filaments in the cells as indicated by an increase in fluorescence. The increase was quantified in the cortex, where it was most apparent, and the fluorescence there was found to increase by about a factor of 3. This increase in cortical phalloidin fluorescence was inhibited by latrunculin B at the same concentration required to inhibit Ca(2+) gradient formation and photopolarization. The distribution of the increasing phalloidin fluorescence was uniform with respect to the developing rhizoid-thallus axis during the formation of the axis, and no intense patches of fluorescence were observed. After germination, fluorescence suggestive of an apical ring of actin filaments was seen near the rhizoid tip. Finally, inhibitor studies indicated that myosin may be involved in the photopolarization process.     

The spindle: a dynamic assembly of microtubules and motors

In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.     

Kinesin-5 is not essential for mitotic spindle elongation in Dictyostelium

The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13(-) cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves in kif13(-) cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division.     

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.     

Blue- and green-light signals for gamete release in the brown alga, Silvetia compressa

The intertidal brown alga Silvetia compressa releases gametes from receptacles (the reproductive tissue) rapidly upon a dark transfer (following a photosynthesis-dependent period in the light, termed potentiation). In this study, the wavelength-dependence of this process was investigated. During the potentiation period in white light (WL), gametes are not released. However, gametes were released during potentiation in blue light (BL), or in low red light/blue light (RL/BL) ratios, but not in RL alone, high RL/BL ratios, or in broadband blue-green light (B-GL) (presence of BL, but absence of RL). RL was as effective as WL for potentiation, i.e., both lead to gamete release following transfer to darkness. Rates of linear photosynthetic electron transport were similar in RL and BL. Gamete release in BL was inhibited by equal amounts of additional narrow-waveband light between the green and red regions of the spectrum, with light-induced gamete release restricted between <491 nm and 509 nm. Very little light-induced gamete release occurred between 530 nm and 650 nm. It is proposed that a BL-responsive photoreceptor is responsible for light-induced gamete release. Transfer of WL-potentiated receptacles to GL near 530 nm resulted in significant de-potentiation and reduced gamete release during a subsequent dark transfer. This effect was not seen at 509 nm or 560 nm and revealed the presence of a second photoreceptor system repressing or counteracting potentiation in the light. We propose that the restriction of gamete release to periods when irradiance is blue-shifted may constitute a depth-sensing mechanism for this intertidal alga, allowing controlled release of gametes at high tide and/or less turbid periods, thus minimizing gamete dilution, and promoting fertilization success.     

Roles and mechanisms of Kinesin-6 KIF20A in spindle organization during cell division

Mitotic kinesin is crucial for spindle assembly and chromosome segregation in cell division. KIF20A/MKlp2, a member of kinesin-6 subfamily, plays important roles in the central spindle organization at anaphase and cytokinesis. In this review, we briefly introduce the discovery and classification of kinesin-6 motors in model organisms, and summarize the biochemical features and mechanics of KIF20A proteins. We emphasize the complicated interactions of KIF20A with partner proteins, including MKlp1, Plk1 and Rab6. Particularly, we highlight the regulation of Cdk1 and chromosomal passenger complex on kinesin-6 KIF20A at late stage of mitosis. We summarized the multiple functions of KIF20A in central spindle assembly and the formation of cleavage furrow in both mitosis and meiosis. In addition, we conclude the expression patterns of KIF20A in tumorigenesis and its applications in tumor therapy.     

Acetylated microtubules are required for fusion of autophagosomes with lysosomes

Background  

Autophagy is a dynamic process during which isolation membranes package substrates to form autophagosomes that are fused with lysosomes to form autolysosomes for degradation. Although it is agreed that the LC3II-associated mature autophagosomes move along microtubular tracks, it is still in dispute if the conversion of LC3I to LC3II before autophagosomes are fully mature and subsequent fusion of mature autophagosomes with lysosomes require microtubules.     

Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.     

NuMA is required for the organization of microtubules into aster-like mitotic arrays

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.     

Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

Dynamics of spindle microtubule organization: kinetochore fiber microtubules of plant endosperm

Organization of kinetochore fiber microtubules (MTs) throughout mitosis in the endosperm of Haemanthus katherinae Bak. has been analysed using serial section reconstruction from electron micrographs. Accurate and complete studies have required careful analysis of individual MTs in precisely oriented serial sections through many (45) preselected cells. Kinetochore MTs (kMTs) and non-kinetochore MTs (nkMTs) intermingle within the fiber throughout division, undergoing characteristic, time- dependent, organizational changes. The number of kMTs increases progressively throughout the kinetochore during prometaphase-metaphase. Prometaphase chromosomes which were probably moving toward the pole at the time of fixation have unequally developed kinetochores associated with many nkMTs. The greatest numbers of kMTs (74-109/kinetochore), kinetochore cross-sectional area, and kMT central density all occur at metaphase. Throughout anaphase and telophase there is a decrease in the number of kMTs and, in the kinetochore cross-sectional area, an increased obliquity of kMTs and increased numbers of short MTs near the kinetochore. Delayed kinetochores possess more kMTs than do kinetochores near the poles, but fewer kMTs than chromosomes which have moved equivalent distances in other cells. The frequency of C-shaped proximal MT terminations within kinetochores is highest at early prometaphase and midtelophase, falling to zero at midanaphase. Therefore, in Haemanthus, MTs are probably lost from the periphery of the kinetochore during anaphase in a manner which is related to both time and position of the chromosome along the spindle axis. The complex, time-dependent organization of MTs in the kinetochore region strongly suggests that chromosome movement is accompanied by continual MT rearrangement and/or assembly/disassembly.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.     

Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization

Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.     

Human Nek7-interactor RGS2 is required for mitotic spindle organization

The mitotic spindle apparatus is composed of microtubule (MT) networks attached to kinetochores organized from 2 centrosomes (a.k.a. spindle poles). In addition to this central spindle apparatus, astral MTs assemble at the mitotic spindle pole and attach to the cell cortex to ensure appropriate spindle orientation. We propose that cell cycle-related kinase, Nek7, and its novel interacting protein RGS2, are involved in mitosis regulation and spindle formation. We found that RGS2 localizes to the mitotic spindle in a Nek7-dependent manner, and along with Nek7 contributes to spindle morphology and mitotic spindle pole integrity. RGS2-depletion leads to a mitotic-delay and severe defects in the chromosomes alignment and congression. Importantly, RGS2 or Nek7 depletion or even overexpression of wild-type or kinase-dead Nek7, reduced γ-tubulin from the mitotic spindle poles. In addition to causing a mitotic delay, RGS2 depletion induced mitotic spindle misorientation coinciding with astral MT-reduction. We propose that these phenotypes directly contribute to a failure in mitotic spindle alignment to the substratum. In conclusion, we suggest a molecular mechanism whereupon Nek7 and RGS2 may act cooperatively to ensure proper mitotic spindle organization.     

HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous polyglutamine aggregates via a process that appears to involve capture and degradation of aggregates by autophagy. The ubiquitin-proteasome system protects cells against proteotoxicity by degrading soluble monomeric misfolded aggregation-prone proteins but is ineffective against, and impaired by, non-native protein oligomers. Here we show that autophagy is induced in response to impaired ubiquitin proteasome system activity. We show that ATG proteins, molecular determinants of autophagic vacuole formation, and lysosomes are recruited to pericentriolar cytoplasmic inclusion bodies by a process requiring an intact microtubule cytoskeleton and the cytoplasmic deacetylase HDAC6. These data suggest that HDAC6-dependent retrograde transport on microtubules is used by cells to increase the efficiency and selectivity of autophagic degradation.