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

Abstract

Titin, the giant elastic protein found in muscles, is present in spindles of crane-fly and locust spermatocytes as determined by immunofluorescence staining using three antibodies, each raised against a different, spatially separated fragment of Drosophila titin (D-titin). All three antibodies stained the Z-lines and other regions in insect myofibrils. In western blots of insect muscle extract the antibodies reacted with high molecular mass proteins, ranging between rat nebulin (600-900 kDa) and rat titin (3000-4000 kDa). Mass spectrometry of the high molecular mass band from the Coomassie-Blue-stained gel of insect muscle proteins indicates that the protein the antibodies bind to is titin. The pattern of staining in insect spermatocytes was slightly different in the two species, but in general all three anti-D-titin antibodies stained the same components: the chromosomes, prophase and telophase nuclear membranes, the spindle in general, along kinetochore and non-kinetochore microtubules, along apparent connections between partner half-bivalents during anaphase, and various cytoplasmic components, including the contractile ring. That the same cellular components are stained in close proximity by the three different antibodies, each against a different region of D-titin, is strong evidence that the three antibodies identify a titin-like protein in insect spindles, which we identified by mass spectrometry analysis as being titin. The spindle matrix proteins skeletor, megator and chromator are present in many of the same structures, in positions very close to (or the same as) D-titin. Myosin and actin also are present in spindles in close proximity to D-titin. The varying spatial arrangements of these proteins during the course of division suggest that they interact to form a spindle matrix with elastic properties provided by a titin-like protein.

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.…”

Latrunculin A Delays Anaphase Onset in Fission Yeast by Disrupting an Ase1-independent Pathway Controlling Mitotic Spindle Stability

“…An actin meshwork aids chromosome congression during the first meiotic division of starfish oocytes, but this is thought to be necessary only because microtubule-based kinetochore capture is inefficient in large cells (Lenart et al, 2005). Evidence for an actin-based spindle matrix has also been reported in crane fly spermato- cytes, and this is thought to act in conjunction with other spindle matrix proteins, such as Chromator, Skeletor, and Megator (Silverman-Gavrila and Forer, 2000;Fabian et al, 2007). In budding yeast, Fin1p forms filaments between the separated spindle poles during mitosis, and it is essential for viability in the absence of Ase1p (van Hemert et al, 2002;Woodbury and Morgan, 2007).…”

An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells

“…However, Axs appeared to be absent from the internal spindle area, but rather distributed in a sheath-like layer enclosing the meiotic spindle in Drosophila (Kramer and Hawley, 2003). Recently titin, a gigantic elastic protein normally involved in muscle contraction was identified in the spindle area of Caenorhabditis elegans cells (Zastrow et al, 2006) and in insect spermatocytes (Fabian et al, 2007).…”