Structure of kinetochore fibres in crane‐fly spermatocytes after irradiation with an ultraviolet microbeam: Neither microtubules nor actin filaments remain in the irradiated region

Abstract

We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving. We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy. For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation. Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions. Similarly, actin filaments normally present in kinetochore fibres were severed by the ultraviolet microbeam irradiations: the irradiated regions contained no actin filaments and only local spots of non-filamentous actin. There was no difference in irradiated regions when the associated chromosomes continued to move versus when they stopped moving. Thus, one cannot explain motion with severed kinetochore microtubules in terms of either microtubules or actin-filaments bridging the irradiated region. The data seem to negate current models for anaphase chromosome movement and support a model in which poleward chromosome movement results from forces generated within the spindle matrix that propel kinetochore fibres or kinetochore stubs poleward.

Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

“…Abundant evidence has accumulated that actin and myosin (including activated (phosphorylated) myosin) are present in spindles in general and kinetochore fibers in particular (reviews in Forer et al 2003;Woolner and Bement 2009;Dulyaninova et al 2004;Weber et al 2004;Fabian et al 2007b;Fabian and Forer 2007;Woolner et al 2008;Vilmos et al 2009), as are proteins that interact in actin-myosin function such as titin (Fabian et al 2007a), Band 4.1 (Krauss et al 1997), zyxin (Hirota et al 2000), myosin light chain kinase (Dulyaninova et al 2004), and moesin (Vilmos et al 2009). Actin and myosin interact with microtubules in a variety of other motile situations (e.g., Rodriguez et al 2003;Weber et al 2004;Pizon et al 2005;Woolner and Bement 2009) so it would not be surprising were they to interact with spindle microtubules.…”

“…Actin and myosin interact with microtubules in a variety of other motile situations (e.g., Rodriguez et al 2003;Weber et al 2004;Pizon et al 2005;Woolner and Bement 2009) so it would not be surprising were they to interact with spindle microtubules. Not only are they present in spindles but a variety of experimental perturbations has also implicated them in spindle function (reviewed in Forer et al 2003;Pickett-Heaps and Forer 2009;Woolner and Bement 2009;Ma et al 2010). For example, interfering with actin and/or myosin alters anaphase chromosome movement, blocks anaphase onset, and alters spindle shapes, chromosome attachment, and chromosome distributions.…”

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane‐fly spermatocytes

“…Actin/myosin involvement in mitosis has been deduced from studies that actin, myosin and titin are present in a variety of spindles [e.g., Table I of Forer et al, 2003;Robinson and Snyder, 2005;Yasuda et al, 2005;Fabian and Forer, 2007;Fabian et al, 2007b] and, also in a variety of cells, from studies of the effects of actin inhibitors, of myosin inhibitors or of genetic alterations to myosin [review in Table II of ; also Forer, 2005, 2007;Woolner et al, 2008]. In crane-fly spermatocytes in particular, the cells used for the experiments we describe herein, anaphase chromosome movement is altered by actin inhibitors cytochalasin D and latrunculin B, both of which cause depolymerization of actin filaments, and is altered by myosin inhibitors BDM and Y27632, both of which block myosin activity [Forer and Pickett-Heaps, 1998;Silverman-Gavrila and Forer, 2001;Fabian and Forer, 2005;Fabian et al, 2007a].…”

Titin in insect spermatocyte spindle fibers associates with microtubules, actin, myosin and the matrix proteins skeletor, megator and chromator

“…Furthermore, an elastic spindle matrix, in conjunction with other spindle forces, might be responsible both for the wobbling motion of the kinetochore stub and for the continued poleward motion of the chromosome after a UV microbeam severs a single kinetochore fiber in anaphase cranefly spermatocytes (Pickett-Heaps et al, 1996;Forer et al, 2003). Direct evidence of a spindle matrix was provided by isolation of a protein (skeletor) from Drosophila cells.…”