Some retrospectives on early studies of plant microtubules.

We pay tribute to the seminal paper 'A microtubule in plant cell fine structure' by Myron C. Ledbetter and Keith R. Porter (1963) by summarizing the very limited knowledge of plant cell ultrastructure that we had prior to that publication, and, by way of our three retrospective accounts, show how this paper stimulated and influenced subsequent research on plant microtubules.

Precocious (pre-anaphase) cleavage furrows in Mesostoma spermatocytes.

It generally is assumed that cleavage furrows start ingression at anaphase, but this is not always true. Cleavage furrows are initiated during prometaphase in spermatocytes of the flatworm Mesostoma, becoming detectable soon after the spindles achieve bipolarity. The furrows deepen during prometaphase, but ingression soon arrests.

MICROMORPHOGENESIS DURING DIATOM WALL FORMATION PRODUCES SILICEOUS NANOSTRUCTURES WITH DIFFERENT PROPERTIES(1).

Micromorphogenesis within the silica deposition vesicle (SDV) of the diatom Pinnularia viridis (Nitzsh) Ehrenb. resulted in distinct silica nanostructures and layers within forming valves and girdle bands. These siliceous components were similarly disclosed following alkaline etching of mature valves/girdle bands, where their different susceptibilities to dissolution over time resulted from apparent differences in silica density and/or chemistry.

Mitosis: spindle evolution and the matrix model.

Current spindle models explain "anaphase A" (movement of chromosomes to the poles) in terms of a motility system based solely on microtubules (MTs) and that functions in a manner unique to mitosis. We find both these propositions unlikely. An evolutionary perspective suggests that when the spindle evolved, it should have come to share not only components (e.

What generates flux of tubulin in kinetochore microtubules?

We discuss models for production of tubulin flux in kinetochore microtubules. Current models concentrate solely on microtubules and their associated motors and enzymes. For example, in some models the driving force for flux is enzymes at the poles and the kinetochores; in others the driving force is motor molecules that are associated with a stationary spindle matrix.

Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes.

We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the "kinetochore stubs", the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules.

Fibrin clots keep non-adhering living cells in place on glass for perfusion or fixation.

We describe a method to hold living cells in place that ordinarily do not adhere to glass coverslips. The method, developed for insect spermatocytes but with application to other cell types, consists of embedding cells in a fibrin clot that forms after the enzyme thrombin cleaves the blood protein fibrinogen. The method permits continuous observation of living cells as they are treated with and recover from drug or other treatments: when held in the clot the living cells remain in place and keep their shapes when perfused with drugs that ordinarily cause drastic shape changes, and they remain in place and keep their shapes through lysis/fixation procedures.

Spatial determinants in morphogenesis: recovery from plasmolysis in the diatom Ditylum.

Ditylum cells are enclosed in a rigid wall consisting of two "valves" (end walls) connected by "girdle bands." A hollow spine, the Labiate Process (LP), extends from each valve and a stable cytoplasmic strand connects its base with the nucleus. We investigated whether cells might possess "spatial determinants" for controlling their internal organization and wall morphogenesis.

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

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.

Phallacidin stains the kinetochore region in the mitotic spindle of the green algae Oedogonium spp.

We found previously that in living cells of Oedogonium cardiacum and O. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin.

Pac-Man does not resolve the enduring problem of anaphase chromosome movement.

The Pac-Man hypothesis suggests that poleward movement of chromosomes during anaphase A is brought about by: disassembly of kinetochore microtubules (MTs) at the kinetochore; generation of the poleward force exclusively at or very close to the kinetochore; and the required energy coming from coupled disassembly of these MTs. This model has become widely accepted and cited as the sole or major mechanism of anaphase A. Rarely acknowledged are several significant phenomena that refute some or all of these postulates.

The cytoplast concept in dividing plant cells: cytoplasmic domains and the evolution of spatially organized cell.

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single "tensegral" unit. The term "tensegrity" is a contraction of "tensional integrity" and the concept proposes that the whole cell is organized by an integrated cytoskeleton of tension elements (e.

Cytochalasin D and latrunculin affect chromosome behaviour during meiosis in crane-fly spermatocytes.

Living crane-fly spermatocytes were treated with 10-20 microg/ml cytochalasin D (CD) or 0.3 microg/ml latrunculin (LAT) at various stages of meiosis I. The drugs had the same effects on chromosome behaviour, but CD effects were reversible and LAT effects generally were not.

Traction fibre: toward a "tensegral" model of the spindle.

Most current hypotheses of mitotic mechanisms are based on the "PAC-MAN" paradigm in which chromosome movement is generated and powered by disassembly of kinetochore microtubules (k-MTs) by the kinetochore. Recent experiments demonstrate that this model cannot explain force generation for anaphase chromosome movement [Pickett-Heaps et al., 1996: Protoplasma 192:1-10].

Ultraviolet microbeam irradiations of epithelial and spermatocyte spindles suggest that forces act on the kinetochore fibre and are not generated by its disassembly.

Ultraviolet (UV) microbeam irradiations of crane-fly spermatocyte and newt epithelial spindles severed kinetochore fibres (KT-fibres), creating areas of reduced birefringence (ARBs): the remnant KT-fibre consists of two "stubs," a pole-stub attached to the pole and a KT-stub attached to the kinetochore. KT-stubs remained visible but pole-stubs soon became undetectable [Forer et al., 1996].

The amyloid precursor protein of Alzheimer's disease is found on the surface of static but not activity motile portions of neurites.

We have previously found that the amyloid precursor protein (APP) of Alzheimer's disease is present on the surface of rat cortical neurons in culture, in a segmental pattern which first becomes evident after 24 hours and is fully developed by five days. As APP has previously been reported to have a short half-life in neuronal cell lines, and has been shown to contain binding sites for various extracellular matrix components within its extracellular domain, we hypothesized that APP would be associated with portions of neurites undergoing rapid structural change, such as growth cones. To test this hypothesis, we observed selected neurons by video time-lapse differential interference microscopy on 24-hour-old primary rat neuronal cultures for up to 45 minutes, followed by fixation and immunocytochemistry to ascertain surface APP distribution on those same neurons.

The effects of diazepam on mitosis and the microtubule cytoskeleton. I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta.

The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy. DZP treatment (250 microM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle.

An extended corona attached to metaphase kinetochores of the green alga Oedogonium.

Mitotic cells of the green alga Oedogonium were treated with the anti-microtubule agent oryzalin (1.0-0.1 microM) for 5 to 10 min.

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region.

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation.

The effects of colchicine and dinitrophenol on the in vivo rates of anaphase A and B in the diatom Surirella.

The rates of chromosome-to-pole movement (anaphase A) and pole-pole separation (anaphase B) in vivo were measured in the pennate diatom Surirella, using differential interference contrast (DIC) light microscopy. In control cells, the rate of anaphase A is 1.6 +/- 0.

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened.

Microtubule dynamics in the spindle. Theoretical aspects of assembly/disassembly reactions in vivo.

The mitotic spindle contains several classes of microtubules (MTs) whose lengths change independently during mitosis. Precise control over MT polymerization and depolymerization during spindle formation, anaphase chromosome movements, and spindle breakdown is necessary for successful cell division. This model proposes the site of addition and removal of MT subunits in each of four classes of spindle MTs at different stages of mitosis, and suggests how this addition and removal is controlled.