Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast.

The current model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by its assembly factor Mdm36.

An Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein.

In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by SDS-PAGE analysis of the supernatant and pellet fractions. However, MAPs that form large oligomers tend to pellet on their own during the centrifugation step, making it difficult to assess co-sedimentation.

Num1 versus NuMA: insights from two functionally homologous proteins.

In both animals and fungi, spindle positioning is dependent upon pulling forces generated by cortically anchored dynein. In animals, cortical anchoring is accomplished by a ternary complex containing the dynein-binding protein NuMA and its cortical attachment machinery. The same function is accomplished by Num1 in budding yeast.

Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1.

Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In , the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function.

Microtubule cross-linking activity of She1 ensures spindle stability for spindle positioning.

Dynein mediates spindle positioning in budding yeast by pulling on astral microtubules (MTs) from the cell cortex. The MT-associated protein She1 regulates dynein activity along astral MTs and directs spindle movements toward the bud cell. In addition to localizing to astral MTs, She1 also targets to the spindle, but its role on the spindle remains unknown.

Cytoskeletal dynamics: a view from the membrane.

Many aspects of cytoskeletal assembly and dynamics can be recapitulated in vitro; yet, how the cytoskeleton integrates signals in vivo across cellular membranes is far less understood. Recent work has demonstrated that the membrane alone, or through membrane-associated proteins, can effect dynamic changes to the cytoskeleton, thereby impacting cell physiology. Having identified mechanistic links between membranes and the actin, microtubule, and septin cytoskeletons, these studies highlight the membrane's central role in coordinating these cytoskeletal systems to carry out essential processes, such as endocytosis, spindle positioning, and cellular compartmentalization.

Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.

The ability to fluorescently label microtubules in live cells has enabled numerous studies of motile and mitotic processes. Such studies are particularly useful in budding yeast owing to the ease with which they can be genetically manipulated and imaged by live cell fluorescence microscopy. Because of problems associated with fusing genes encoding fluorescent proteins (FPs) to the native α-tubulin (TUB1) gene, the FP-Tub1 fusion is generally integrated into the genome such that the endogenous TUB1 locus is left intact.

The role of +TIPs in directional tip expansion.

Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron-like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth - the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs.

Microtubule motors: doin' it without dynactin.

The minus-end directed microtubule motor protein cytoplasmic dynein contributes to many aspects of cell division and it is generally believed that these mitotic functions require the dynein activator and processivity factor, dynactin. New research now shows that dynein accomplishes many of its mitotic functions without dynactin.

She1-mediated inhibition of dynein motility along astral microtubules promotes polarized spindle movements.

Background: Cytoplasmic dynein motility along microtubules is critical for diverse cellular processes ranging from vesicular transport to nuclear envelope breakdown to mitotic spindle alignment. In yeast, we have proposed a regulated-offloading model to explain how dynein motility drives microtubule sliding along the cortex, powering transport of the nucleus into the mother-bud neck [1, 2]: the dynein regulator She1 limits dynein offloading by gating the recruitment of dynactin to the astral microtubule plus end, a prerequisite for offloading to the cortex. However, whether She1 subsequently affects cortically anchored dynein activity during microtubule sliding is unclear.

Cell cycle-regulated cortical dynein/dynactin promotes symmetric cell division by differential pole motion in anaphase.

In cultured mammalian cells, how dynein/dynactin contributes to spindle positioning is poorly understood. To assess the role of cortical dynein/dynactin in this process, we generated mammalian cell lines expressing localization and affinity purification (LAP)-tagged dynein/dynactin subunits from bacterial artificial chromosomes and observed asymmetric cortical localization of dynein and dynactin during mitosis. In cells with asymmetrically positioned spindles, dynein and dynactin were both enriched at the cortex distal to the spindle.

Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation.

During animal development, microtubules (MTs) play a major role in directing cellular and subcellular patterning, impacting cell polarization and subcellular organization, thereby affecting cell fate determination and tissue architecture. In particular, when progenitor cells divide asymmetrically along an anterior-posterior or apical-basal axis, MTs must coordinate the position of the mitotic spindle with the site of cell division to ensure normal distribution of cell fate determinants and equal sequestration of genetic material into the two daughter cells. Emerging data from diverse model systems have led to the prevailing view that, during mitotic spindle positioning, polarity cues at the cell cortex signal for the recruitment of NuMA and the minus-end directed MT motor cytoplasmic dynein.

Astral microtubule asymmetry provides directional cues for spindle positioning in budding yeast.

Cortical force generators play a central role in the orientation and positioning of the mitotic spindle. In higher eukaryotes, asymmetrically localized cortical polarity determinants recruit or activate such force generators, which, through interactions with astral microtubules, position the mitotic spindle at the future site of cytokinesis. Recent studies in budding yeast have shown that, rather than the cell cortex, the astral microtubules themselves may provide polarity cues that are needed for asymmetric pulling on the mitotic spindle.

A novel patch assembly domain in Num1 mediates dynein anchoring at the cortex during spindle positioning.

During mitosis in budding yeast, cortically anchored dynein generates pulling forces on astral microtubules to position the mitotic spindle across the mother-bud neck. The attachment molecule Num1 is required for dynein anchoring at the cell membrane, but how Num1 assembles into stationary cortical patches and interacts with dynein is unknown. We show that an N-terminal Bin/Amphiphysin/Rvs (BAR)-like domain in Num1 mediates the assembly of morphologically distinct patches and its interaction with dynein for spindle translocation into the bud.

Regulated offloading of cytoplasmic dynein from microtubule plus ends to the cortex.

Cytoplasmic dynein mediates spindle orientation from the cell cortex through interactions with astral microtubules, but neither the mechanism governing its cortical targeting nor the regulation thereof is well understood. Here we show that yeast dynein offloads from microtubule plus ends to the daughter cell cortex. Mutants with an engineered peptide inserted between the tail domain and the motor head retain wild-type motor activity but exhibit enhanced offloading and cortical targeting.

Quantitative analysis of Pac1/LIS1-mediated dynein targeting: Implications for regulation of dynein activity in budding yeast.

LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either.

Variations on theme: spindle assembly in diverse cells.

The mitotic spindle faithfully separates the genetic material, and has been reverently observed for well over a century. Across eukaryotes, while the mechanisms for moving chromosomes seem quite conserved, mechanisms for assembling the spindle often seem distinct. Two major pathways for spindle assembly are known, one based on centrosomes and the other based on chromatin, and these pathways are usually considered to be fundamentally different.

Imaging protein dynamics in live mitotic cells.

To ensure that genetic material is accurately segregated during mitosis, eukaryotic cells assemble a mitotic spindle, a dynamic structure composed of microtubules and associated regulatory, structural and motor proteins. Although much has been learned in the past decades from direct observations of live cells expressing fluorescently tagged spindle proteins, a complete understanding of spindle assembly requires a detailed analysis of the dynamic behavior of component parts. Proteins tagged with conventional fluorophores, however, make such an analysis difficult because all of the molecules are uniformly fluorescent.

A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment.

During mitosis in budding yeast, cortically anchored dynein exerts pulling forces on cytoplasmic microtubules, moving the mitotic spindle into the mother-bud neck. Anchoring of dynein requires the cortical patch protein Num1, which is hypothesized to interact with PI(4,5)P(2) via its C-terminal pleckstrin homology (PH) domain. Here we show that the PH domain and PI(4,5)P(2) are required for the cortical localization of Num1, but are not sufficient to mediate the cortical assembly of Num1 patches.

Motor- and tail-dependent targeting of dynein to microtubule plus ends and the cell cortex.

Background: Cytoplasmic dynein mediates spindle positioning in budding yeast by powering sliding of microtubules along the cell cortex. Although previous studies have demonstrated cortical and plus-end targeting of dynein heavy chain (Dyn1/HC), the regulation of its recruitment to these sites remains elusive.

Results: Here we show that separate domains of Dyn1/HC confer differential localization to the dynein complex.

Photoactivatable GFP tagging cassettes for protein-tracking studies in the budding yeast Saccharomyces cerevisiae.

Yeast cell biologists use a variety of fluorescent protein tags for determining protein localization and for measuring protein dynamics using fluorescence recovery after photobleaching (FRAP). Although many modern fluorescent proteins, such as those with photoactivatable and photoconvertible characteristics, have been developed, none has been exploited for studies in budding yeast. We describe here the construction of yeast-tagging vectors containing photoactivatable green fluorescent protein (PA-GFP) for analysis of protein behaviour.

NudEL targets dynein to microtubule ends through LIS1.

Dynein is a minus-end-directed microtubule motor with critical roles in mitosis, membrane transport and intracellular transport. Several proteins regulate dynein activity, including dynactin, LIS1 (refs 2, 3) and NudEL (NudE-like). Here, we identify a NUDEL homologue in budding yeast and name it Ndl1.

The offloading model for dynein function: differential function of motor subunits.

During mitosis in budding yeast, dynein moves the mitotic spindle into the mother-bud neck. We have proposed an offloading model to explain how dynein works. Dynein is targeted to the dynamic plus end of a cytoplasmic microtubule, offloads to the cortex, becomes anchored and activated, and then pulls on the microtubule.

The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast.

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway.