Andrew S. Bajer

Formation of spindle fibers, kinetochore orientation, and behavior of the nuclear envelope during mitosis in endosperm

The formation of kinetochore (chromosomal) and continuous fibers, and the behavior of the nuclear envelope (NE) was described in studies combining light and electron microscopy. Microtubules (MTs) “push” and “pull” the NE which becomes progressively weaker before breaking. It breaks to a certain extent due to mechanical pressure. Clear zone MTs penetrate into the nuclear area as dense bundles and form continuous fibers. These MTs also attach to some kinetochores during this process. Some kinetochore fibers seem to be formed by the kinetochores themselves which are also responsible for further development and changes of kinetochore fibers. Formation of kinetochore fibers is asynchronous for different chromosomes and even for two sister kinetochores. Often temporary “faulty” connections between different kinetochores or the polar regions are formed which usually break in later stages. This results in movements of chromosomes toward the poles and across the spindle during prometaphase. The NE, whose fine structure has been described, breaks into small pieces which often persist to the next mitosis. Old pieces of NE are utilized in the formation of new NE at telophase. Several problems concerning the mechanism of chromosome movements, visibility of the NE, etc., have also been discussed.

Behavior and fine structure of spindle fibers during mitosis in endosperm

Endosperm ofHaemanthus katherinae has been used as material. Changes in arrangement of spindle fibers, their movements, and behavior of substructures as seen in living cells with the Nomarski system are described. The same cell has been observed with the light microscope and subsequently after the usual procedures with the electron microscope. Arrangement of microtubules forming different types of spindle fibers and their relation to each other during the progress of mitosis is described. Kinetochore structure has also been studied. It is suggested that kinetochore fibers are transported to the poles during anaphase. This conclusion is supported by fine structure studies.

Minus end‐directed kinesin‐like motor protein, kcbp, localizes to anaphase spindle poles in Haemanthus endosperm

Microtubule-based motor proteins assemble and reorganize acentrosomal mitotic and meiotic spindles in animal cells. The functions of motor proteins in acentrosomal plant spindles are unknown. The cellulosic cell wall and relative small size of most plant cells precludes accurate detection of the spatial distribution of motors in mitosis. Large cell size and absence of a cellulosic cell wall in Haemanthus endosperm make these cells ideally suited for studies of the spatial distribution of motor proteins during cell division. Immunolocalization of a kinesin-like calmodulin-binding protein (KCBP) in Haemanthus endosperm revealed its mitotic distribution. KCBP appears first in association with the prophase spindle. Highly concentrated within the cores of individual kinetochore fibers, KCBP decorates microtubules of kinetochore-fibers through metaphase. By mid-anaphase (when a barrel-shaped spindle becomes convergent), the protein redistributes and accumulates at the spindle polar regions. In telophase, KCBP relocates toward the phragmoplast and cell plate. These data suggest a role for KCBP in anaphase spindle microtubule convergence, which assures coherence of kinetochore-fibers within each sister chromosome group. Increasing coherence of kinetochore-fibers prevents splitting within each sister chromosome group and formation of multinucleated cells.

Structure and Organization of the Living Mitotic Spindle of Haemanthus Endosperm

New details of mitotic spindle structures in the endosperm of Haemanthus katherinae (Bak) have been demonstrated by differential interference microscopy. Spindle fibers are clearly seen in the living spindle extending from the kinetochores to the polar region. Individual spindle fibers consist of a bundle of smaller filaments which diverge slightly from the kinetochore and intermingle with filaments from other spindle fibers as they approach the polar region. The degree of intermingling increases during metaphase and anaphase. The chromosomes stop moving when the spindle fibers are still 5 to 10microns long; then the fibers disappear. These observations explain some aspects of spindle movements which were difficult to reconcile with earlier concepts of spindle organization.

Fine structure studies on phragmoplast and cell plate formation

Formation and development of phragmoplast and cell plate were studied in endosperm of Haemanthus katherinaeBak. The same cells were studied with the light and electron microscope. Several cells were studied with time-lapse microcinematography before fixation. This permitted comparison of structures during their development on both the light and electron microscope level. Movements of fibrillar components of the phragmoplast and spindle were analyzed and their transport properties were correlated with formation of the cell plate. Change of arrangement of microtubules, transport of vesicles which form the cell plate, and formation of vesicles and microtubules has also been discussed.

Dynamics of spindle fibers and microtubules during anaphase and phragmoplast formation

Detailed correlation of in vitro observations with the arrangement of microtubules (MTs) during anaphase-telophase were made on endosperm of Haemanthus katherinae. It is stressed that the general course of events leading to the formation of the phragmoplast is the same in all cells, but considerable variation of details may be found in different objects and even in various cells of the same tissue. The changes of MT arrangement in the interzonal region responsible for formation of the phragmoplast already occur in anaphase. During this stage continuous fibers (composed of numerous MTs) lengthen, become thinner (the number of MTs on a cross-section decreases), and often seem to break. After mid-anaphase, thin fibers begin to oscillate transversely to the axis of the phragmoplast and often are considerably laterally displaced (lateral movements). The longest MTs in the phragmoplast are present during oscillations and lateral movements. The new MTs arise in the phragmoplast regions depleted of MTs as a result of lateral movements (usually geometric central region of the phragmoplast). Clusters of vesicles, which accumulate in relation to MTs which move, fuse and form the cell plate. After the fusion, the number and the length of MTs decrease. Several processes are superimposed and occur simultaneously. Also the cell plate is, as a rule, in different stages of development in various regions of the phragmoplast. The movements of MTs and fusion of the vesicles is complex and the details of these processes are not entirely clear. The data supplied here modify some generally accepted concepts of phragmoplast formation and development. This concerns the center of origin of new MTs, the moment when they arise, and the way they subsequently behave.

Relation of F-actin Organization to Microtubules in Drug Treated Haemanthus Mitosis

We report observations on the effects of cytochalasin D, pressure injection of anti-actin antibody, and of a microtubule-disrupting drug (amiprophos methyl), on the distribution of F-actin bundles in endosperm of the higher plant, Haemanthus. The course of mitosis and cell plate formation in vivo was recorded with video time-lapse. Cells recorded in vivo were fixed and processed with non-fading immunogold and/or immunogold silver-enhanced stain to follow the distribution of actin bundles or microtubules. Double stain (actin and microtubules in the same cell) was applied extensively. Three-dimensional spatial relation of actin to microtubules was analyzed with serial optical sectioning in video microscopy.

Morphological Aspects of normal and Abnormal Mitosis

More than one-hundred years of studies on mitosis have resulted in the accumulation of an enormous amount of facts. The early period of study has been reviewed by Wilson [55] and some general ideas concerning attempts of interpretation of previously observed phenomena have been cited by Schrader [51]. Mazia [38] presents a synthesis of our current knowledge.

Meiosis in Cepaea nemoralis studied by microcinematography

SummarySome aspects of meiosis in spermiocytes of Cepaea nemoralis L. are presented. They are based on in vitro studies with the use of time-lapse 16 mm film recording. The main results are:1.The nucleus changes its shape irregularly during diakinesis (and probably earlier stages) until the disappearance of the nuclear membrane. During this period the prophase bivalents inside the nucleus change their position and shape irregularly. Suggestions concerning the importance of such movements are made.2.In many spermiocyte divisions the spindle oscillates regularly in one or several planes. In all cells the periodicity is of more or less the same duration, about 2 mins.3.The volume of the nucleus diminishes during the 1–2 hour period before the disappearance of the nuclear membrane and increases again rapidly a few minutes before the nuclear membrane disappears. The disappearance of the nuclear membrane is followed by a rapid condensation of the space occupied by the chromosomes (contraction stage). This space increases again during prometaphase. The latter events closely resemble those in mitosis of plant endosperm cells.

Reorganization of microtubular cytoskeleton and formation of cellular processes during post-telophase in Haemanthus endosperm

We followed time-dependent post-telophase reorganization of the microtubule cytoskeleton on immunostained preparations of endosperm of the higher plant Haemanthus. After completion of mitosis, the phragmoplast continued to reorganize for several hours. This prompted the formation of phragmoplast-like derivatives (secondary and accessory phragmoplasts and peripheral microtubular ring). Next, elongated cellular protrusions (processes) appeared at the cell periphery. These processes contained long microtubule bundles and disorderly arranged actin filaments. Microtubule converging centers or accessory phragmoplasts were often present at the tips of the processes. Observation in vivo demonstrated that processes were formed at the cell periphery as extensions of lammelipodia or filopodia-type protrusions that commonly terminated with cytoplasmic blobs. We suggest that processes are derivatives of a peripheral microtubular ring that reorganizes gradually into cellular protrusions. Endosperm processes have several features of neuronal cells, or animal somatic cells with overexpressed MAPs. Since microtubule-containing processes were never detected shortly after extrusion of the cells from the embryo sac, this course of events might be restricted specifically to extruded endosperm and triggered either by removal of cells, their placement in monolayer on agar substrate, or both. Thus, post telophase behavior of endosperm cells offers a novel experimental system for studies of cytoskeleton in higher plants.