A cell must build a bipolar mitotic spindle in order to faithfully segregate replicated DNA. To do so, multiple microtubule nucleation pathways are utilized to generate the robust spindle apparatus. TPX2, a microtubule binding protein, holds crucial roles in both the Ran-dependent and Augmin-dependent pathways where microtubules are nucleated near the chromosomes and from pre-existing microtubules. However, the exact role TPX2 plays in branching microtubules is less understood. Here, we explored the effect of truncating the essential TPX2 C-terminal 37 amino acids on Augmin localization and branching microtubule activity. First, we depleted LLC-Pk1 cells of the Augmin subunit HAUS6 and show that microtubule nucleation around the chromosomes following a nocodazole washout is strongly reduced leading to exaggerated kinetochore microtubule growth. Next, we depleted endogenous TPX2 in LLC-Pk1 cells harboring full length or truncated TPX2 bacterial artificial chromosome (BAC) DNA. Results show that TPX2 710 LAP cells have reduced Augmin localization on the spindle fibers, which correlates with reduced microtubule regrowth in the chromosomal region. In TPX2 710 LAP cells, regrowth was like Augmin depleted cells. Therefore, we provide evidence that the far C-terminus of TPX2 is required for branching microtubule nucleation and that kinetochore microtubule growth is Augmin-independent. In addition, we investigated cell cycle regulation of TPX2 by mutating the S738 phosphosite in the C-terminal motor interacting region. We utilized BAC recombineering to create phospho-mimetic and phospho-null mutants. In combination with plasmid DNA knockdown/rescue, overexpression and spindle assembly assays, we show that the phosphorylation of the C-terminal domain contributes to early mitotic events. LLC-Pk1 cells showed a significant increase in aberrant spindle morphology and reduced spindle stability in the presence of 738A and absence of endogenous TPX2. While rescue with the alanine mutant caused in an increase in multipolar spindles, overexpression resulted in a strong dominant negative monopolar phenotype. Therefore, S738 appears to contribute to mitotic force regulation during mitosis. In conclusion, the far C-terminus of TPX2 and its regulation play a role in the formation of a proper mitotic spindle.

No description available.

Cytokinesis separates replicated chromosomes and cytoplasm into two daughter cells. In animal cells, this is achieved by the formation of a cleavage furrow that bisects the mitotic (or meiotic) spindle. It is known that the mitotic apparatus defines the cell cleavage plane. However, it is not clear how the mitotic apparatus initiates the cleavage furrow. Each part of the mitotic apparatus; namely asters, central spindle (microtubule arrays and the spindle midzone), and chromosomes, has been found capable of inducing a cleavage furrow in certain cell types. Yet it is uncertain which part is the essential source of the signal and whether all parts act in concert. This thesis systematically examines in grasshopper spermatocytes 1) which spindle constituent is the essential source of furrow signal; 2) the impact of microtubules on distribution of actin filaments and positioning of cell cleavage relative to spindle reorganization; 3) the independent role of the spindle midzone relative to microtubules in furrow initiation and ingression. These examinations combine micromanipulation with digital-enhanced polarization microscopy and epifluorescence microscopy, in which mitotic spindles in living cells are mechanically dissected and rearranged as desired as well as microfixed to evaluate and propose models for cell cleavage. This thesis has come to the conclusion that none of structural constituents of the spindle apparatus is essential for cell cleavage induction except microtubules. First, furrow induction occurs regardless of a particular spindle constituent, so long as sufficient microtubules are present to form bipolar arrays. Second, microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage. Asymmetric alterations of spindle microtubules dynamically affect the location of the spindle midzone, distribution of actin filaments, and ultimately position of the cleavage furrow in cells containing a bipolar spindle, monopolar spindle, or half-spindle. Third, actin filaments are distributed to the furrow region by microtubule-mediated transport, but organized by the midzone, which is essential for furrow ingression, but not initiation. These results suggest that during post-anaphase spindle assembly, actin filaments are excluded by bipolar microtubule arrays to the equatorial cell cortex where they bundle into a contractile ring with cytokinetic factors. / Graduation date: 2004

The purpose of this study was to determine whether ischemia would enhance muscle spindle responses to tendon tap and vibration during submaximal fatiguing contractions in the soleus muscle of able-bodied individuals. Nine healthy adults attended two experimental sessions approximately 48 hours apart. Both sessions were identical except that the fatigue task in one was performed with a pressure cuff placed above the knee and inflated to 180 mm Hg. Three 5s maximum voluntary contractions (MVCs) were performed prior to and after the fatigue task. Each participant held a target force of 20% MVC until endurance time (peak-to-peak tremor amplitude exceeded 5% MVC or target force dropped by 2% for 3s). Muscle spindle responses were evaluated using the peak-to-peak EMG amplitude of tendon taps (delivered by a custom-made tapper) and the Motor Unit Firing Rates (MUFR) during 15 s of vibration, recorded with fine-wire intramuscular electrodes. H reflex responses were measured before and after fatigue for each condition, to measure the net excitability of the spinal cord. There were no significant differences (p>0.05) in the P-P EMG of tendon taps or the MUFR across any conditions. The post-fatigue Maximal Voluntary Contraction forces were measured and were less than the pre-fatigue values under both conditions (and significantly different in the non-ischemic condition (p=0.01)). Absence of significant differences in the Hmax:Mmax ratios (p=0.94 in non-ischemic/fatigue and p=0.43 in ischemic condition) indicated that the spinal excitability was relatively unchanged across the conditions. Therefore, we could not conclude that ischemia enhanced the muscle spindle response. / text

Muscle spindle fibers are specialized stretch receptors that allow the perception and coordination of limb movement. Differentiation of muscle spindles is initiated by signals derived from the in growing Ia sensory neurons during development. The sensory neuron secretes neuregulin which binds and signals through the ErbB receptors to initiate a signaling cascade. This cascade results in the expression of a specific repertoire of genes, one of which is the transcription factor Egr3, which is necessary in the development of muscle spindles. Signaling occurs efficiently when the postsynaptic receptors are clustered into large aggregates in apposition to an innervating nerve. Using what is known about acetylcholine receptor clustering at neuromuscular junctions as a model, this study shows the importance of the basal lamina proteins agrin and laminin and their shared receptor ??-dystroglycan in aggregating ErbB receptors at sensory synapses. The study also shows that signaling through these receptors subsequently results in increased expression of Egr3, the transcription factor critical to muscle spindle fiber differentiation. Using an ??-dystroglycan silenced culture, it is shown that ??-dystroglycan is necessary to induce neuregulin, laminin and agrin induced Egr3. In these same myotube cultures there is also a reduced number of AChR-ErbB3 colocalized aggregates and this is not rescued with the addition of laminin. Taken together, these results suggest an essential role for basal lamina components and ??-dystroglycan, molecules that are crucial in acetylcholine receptor aggregation at neuromuscular junctions, in the induction of the transcription factor Egr3, a critical transcription factor involved in muscle spindle fiber differentiation.

Signaling systems regulate the flow of cellular information by organizing proteins in space and time to coordinate a variety of cellular activities that are critical for the proper development, function, and maintenance of cells. Signaling molecules can exhibit several levels of complexity through the utilization of modular protein interactions, which can generate simple linear behaviors or complex behaviors such as ultrasensitivity. Protein modularity also serves as the basis for the vast protein networks that form the regulatory networks that govern several biological activities. My work focuses on the importance of protein modularity in complex biological systems, in particular the regulatory pathways of spindle positioning. The first part of my work involves the construction of a synthetic regulatory network using modular protein interactions in an effort to understand the complex behavior of the natural spindle orientation regulator Pins. Utilizing well-characterized protein domains and their binding partners, I built an autoinhibited protein switch that can be activated by a small protein domain. We found that the input-output relationship of the synthetic protein switch could be tuned by the simple addition of "decoy" domains, domains that bind and sequester input signal, thereby impeding the onset of the output response to generate an input threshold. By varying the number and affinities of the decoy domains, we found that we could transform a simple linear response into a complex, ultrasensitive one. Thus, modular protein interactions can serve as a source of complex behaviors. The second part of my work focuses on elucidating the molecular mechanisms underlying spindle positioning in the Drosophila neuroblast. I found that Pins orients the mitotic spindle by coordinating two opposite-polarity microtubule motors Dynein and Kinesin-73 through its multiple domains. Kinesin-73 also relies on its modular domain architecture to perform its duties in Pins-mediated spindle positioning, where its N-terminal half functions in coordinating cortical-microtubule capture while its C-terminal half functions as a region necessary for the activation of Dynein. Thus, modular protein design allows for the organization of spindle orientation regulators in space to achieve the complex biological activity that is spindle positioning. This dissertation includes previously published and unpublished coauthored material. / 10000-01-01

The purpose of this study was to determine the effects of whole-body vibration on electromechanical delay and vertical jump performance. Twenty college aged subjects participated in 10 intervals of whole-body vibration (WBV) at a frequency of 26 Hz and amplitude of 5 mm. Each interval consisted of 60 s of WBV in a half-squat followed by 60 s of rest. After 5 intervals, subjects had 6 min of rest before the final 5 intervals. Each subject also participated in the control which included the same 10 intervals in a half-squat without the WBV. Tests were conducted to assess electromechanical delay (EMD) and vertical jump at baseline, during the 6 min rest period and immediately after the treatment and control. EMD was measured using tibial nerve stimulation and a force plate. EMD was recorded as the lag time between the initiation of gastrocnemeus stimulation and plantar flexion force production. Vertical jump was measured using a force plate and subjects' flight time. The factorial ANOVA results showed no differences between groups, the control and WBV treatment, for both EMD (F (2, 38) = 1.385, p = 0.263) and vertical jump (F (2, 38) = 0.040, p < 0.96). The WBV treatment protocol chosen had no effect on vertical jump. These results suggest that WBV, using this protocol, is not effective for acute vertical jump or EMD enhancement. Also, since there was no effect on EMD, this suggests that the WBV treatment did not enhance muscle spindle sensitivity.

A non-microtubule-based spindle matrix has been observed in Drosophila melanogaster meiotic and somatic cells. This matrix comprises the proteins: Skeletor, Megator and Chromator, which localize to the nuclear periphery and chromosomes at prophase and to the microtubule spindle during mitosis and meiosis. This matrix might play a role in nuclear organization and microtubule assembly and stabilization. It is important to determine the presence or absence of this matrix in other species in order to compare form and function to that in Drosophila. These matrix proteins were studied in both normally dividing cells and cells in which division was disrupted. Our evidence suggests that a spindle matrix exists in meiocytes and embryos of the cricket, Acheta domesticus, and in a mammalian cell line established from Chinese hamster ovary cells. We report the results of our studies on the spindle matrix in invertebrate and mammalian cells and discuss implications of these findings.

No description available.

Oocytes of most species assemble and maintain a functional bipolar spindle in the absence of centrosomes. Strikingly, after bipolar spindle formation, oocytes arrest in metaphase for several hours before fertilisation. How the dynamic spindle maintains its bipolarity during this long arrest is poorly understood. I hypothesise that the bipolar spindle is stably maintained by changes in the distribution of microtubule-associated proteins (MAPs) on the spindle during the long oocyte arrest. To test this, I generated transgenic flies expressing GFP-tagged microtubule-associated proteins (MAPs), and found that 13 out of 24 proteins change localisation between early and late oocytes. I refer to these changes in MAP localisation after establishment of bipolarity as 'spindle maturation'. In order to identify the molecular mechanisms triggering MAP relocalisation, I manipulated the kinase activity of the cell cycle regulator Cdk1 by over-expressing non-degradable cyclin A or B, the major activators of Cdk1. Their expression prevented re-localisation of distinct sets of MAPs, and disrupted spindle bipolarity and accurate chromosome segregation in oocytes. Kinesin-6 Pavarotti/MKlp1 localised strongly to the spindle equator in late oocytes, whilst nearly always absent from this region in early oocytes. The localisation of Pavarotti to the spindle equator in late oocytes was reduced when cyclin B is over-expressed in oocytes, suggesting a role for Cdk1/cyclin B complex in regulating Pavarotti localisation. Indeed, a Pavarotti/Mklp1 mutant non-phosphorylatable by Cdk1 prematurely localised to the meiotic spindle and disrupted spindle bipolarity. Moreover, removal of Pavarotti from the metaphase-I spindle by RNAi induced spindle defects in oocytes. Therefore, it is likely that the microtubule cross-linking activity of Pavarotti enhances the stability of the metaphase-I spindle during the long arrest. Consistent with this, I found that the microtubule density in the spindle equator is higher in late oocytes. Altogether, I propose that remodelling the molecular architecture of the spindle during the long oocyte arrest is important to stabilise the bipolar spindle without centrosomes.