Microtubule Flux Mediates Poleward Motion of Acentric Chromosome Fragments during Meiosis in Insect Spermatocytes

Abstract

We applied a combination of laser microsurgery and quantitative polarization microscopy to study kinetochore-independent forces that act on chromosome arms during meiosis in crane fly spermatocytes. When chromosome arms located within one of the half-spindles during prometaor metaphase were cut with the laser, the acentric fragments (lacking kinetochores) that were generated moved poleward with velocities similar to those of anaphase chromosomes (ϳ0.5 m/min). To determine the mechanism underlying this poleward motion of detached arms, we treated spermatocytes with the microtubule-stabilizing drug taxol. Spindles in taxol-treated cells were noticeably short, yet with polarized light, the distribution and densities of microtubules in domains where fragment movement occurred were not different from those in control cells. When acentric fragments were generated in taxol-treated spermatocytes, 22 of 24 fragments failed to exhibit poleward motion, and the two that did move had velocities attenuated by 80% (to ϳ0.1 m/min). In these cells, taxol did not inhibit the disjunction of chromosomes nor prevent their poleward segregation during anaphase, but the velocity of anaphase was also decreased 80% (ϳ0.1 m/min) relative to untreated controls. Together, these data reveal that microtubule flux exerts pole-directed forces on chromosome arms during meiosis in crane fly spermatocytes and strongly suggest that the mechanism underlying microtubule flux also is used in the anaphase motion of kinetochores in these cells.

A Functional Relationship between NuMA and Kid Is Involved in Both Spindle Organization and Chromosome Alignment in Vertebrate Cells

“…ochore microtubule length, then that force could act as a positional cue to direct chromosome alignment because the spindle equator would be the position where poleward traction forces would be equal between the poles. We favor this model because poleward microtubule flux is a conserved feature of spindles in most cell types (Mitchison, 1989a;Mitchison and Salmon, 1992;Desai et al, 1998;LaFountain et al, 2001;Brust-Mascher and Scholey, 2002;Maddox et al, 2002) and NuMA is localized at spindle poles, the likely position of factors needed to generate poleward microtubule flux (Waters et al, 1996). Furthermore, by examining multivalent chromosome position and kinetochore microtubule lengths in grasshopper spermatocytes, Hays et al (1982) concluded that "the magnitude of traction force on a kinetochore fiber is a linear function of fiber length."…”

Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

“…The general rule that kinetochores lead while chromosome arms follow applies in many cell types including vertebrates [7] and yeasts [8], but there are exceptions, such as in plant endosperm [9], and in crane-fly spermatocytes [10], where arms sometimes lead. These alternative cases remind us that forces are also exerted directly on chromosome arms, although the primary motive forces for anaphase are commonly exerted at kinetochores.…”

Laser Microsurgery in the GFP Era: A Cell Biologist's Perspective

“…In a more biologically successful project, Forer (1965) combined a UV microbeam and polarization light microscopy to show that once severed, spindle (kinetochore) fibers in the cranefly spermatocyte regrew from the chromosome to the spindle pole. These original experiments have since been repeated on more than one occasion, although on different cell types, with increasingly sophisticated UV and then visible laser microbeams (LaFountain, Jr., et al, 2001;Maiato et al, 2004;Spurck et al, 1990).…”

“…In a more biologically successful project, Forer (1965) combined a UV microbeam and polarization light microscopy to show that once severed, spindle (kinetochore) fibers in the cranefly spermatocyte regrew from the chromosome to the spindle pole. These original experiments have since been repeated on more than one occasion, although on different cell types, with increasingly sophisticated UV and then visible laser microbeams (LaFountain, Jr., et al, 2001;Maiato et al, 2004;Spurck et al, 1990).The ability to visualize and thus target for destruction of many intracellular organelles resurrected biologist's interest in micro-photo-surgery. However, it immediately became apparent that there was a major problem of using lampgenerated UV light for this approach: since chromatin (DNA) effciently absorbs 280-nm light, all cellular systems are extremely sensitive to UV, which leads to unavoidable nonspecific (and not always easy to define) side effects (reviewed in Khodjakov et al, 1997b).…”

“…During the next several years this workstation was used to demonstrate, for example, that the spindle assembly checkpoint monitors kinetochore attachment , that chromosomes containing a single kinetochore can congress to the equator of the forming spindle (Khodjakov et al, 1997a), and that entry into mitosis in vertebrate cells is guarded by a DNA damage checkpoint that reverses the cell cycle when triggered during early prophase (Rieder and Cole, 1998). Further, this same instrument was also used by other biologists to address a number of questions in systems as diverse as fungi to cranefly (Inoue et al, 1998;LaFountain, Jr., et al, 2001, 2002Orokos et al, 2000).As emphasized early on by Berns et al (1981), Gaussian laser beams can actually generate a central "hot spot" inside the Airy disk. Thus, it is possible to select a beam energy at which damage to the specimen is restricted only to the "hot spot" at the peak intensity in the center of the Airy disk.…”