Taxol Crystals Can Masquerade as Stabilized Microtubules

Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications

Redistribution of Actin during Assembly and Reassembly of the Contractile Ring in Grasshopper Spermatocytes

Cytokinesis in animal cells requires the assembly of an actomyosin contractile ring to cleave the cell. The ring is highly dynamic; it assembles and disassembles during each cell cleavage, resulting in the recurrent redistribution of actin. To investigate this process in grasshopper spermatocytes, we mechanically manipulated the spindle to induce actin redistribution into ectopic contractile rings, around reassembled lateral spindles. To enhance visualization of actin, we folded the spindle at its equator to convert the remnants of the partially assembled ring into a concentrated source of actin. Filaments from the disintegrating ring aligned along reorganizing spindle microtubules, suggesting that their incorporation into the new ring was mediated by microtubules. We tracked incorporation by speckling actin filaments with Qdots and/or labeling them with Alexa 488-phalloidin. The pattern of movement implied that actin was transported along spindle microtubules, before entering the ring. By double-labeling dividing cells, we imaged actin filaments moving along microtubules near the contractile ring. Together, our findings indicate that in one mechanism of actin redistribution, actin filaments are transported along spindle microtubule tracks in a plus-end–directed fashion. After reaching the spindle midzone, the filaments could be transported laterally to the ring. Notably, actin filaments undergo a dramatic trajectory change as they enter the ring, implying the existence of a pulling force. Two other mechanisms of actin redistribution, cortical flow and de novo assembly, are also present in grasshopper, suggesting that actin converges at the nascent contractile ring from diffuse sources within the cytoplasm and cortex, mediated by spindle microtubules

Dissecting induction of cell cleavage

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

AlsopGBradley2004.pdf

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

Alsop DVD.zip

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