Cine-micrographic studies on mitosis in endosperm

Summary The course of mitosis in endosperm was studied in plants with medium and large chromosomes by the cine-micrographic method; 3 main problems were considered: 1. chromosome movements, 2. cytoplasmic currents, 3. Brownian movements.A short part of the film with whose analysis the present paper deals was projected at the VIII. International Botanical Congress—Paris 1954 as an illustration of the presented communication (Bajer 1954 b).These studies have been made possible thanks to a grant from the Polish Academy of Sciences, which is gratefully acknowledged.     

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.     

Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline

The distribution of membrane-associated calcium has been determined at various stages of mitosis in Haemanthus endosperm cells with the fluorescent chelate probe chlorotetracycline (CTC). CTC fluorescence in Haemanthus has two components: punctate, because of mitochondrial and plastid membrane-Ca++; and diffuse, primarily because of Ca++ associated with endoplasmic reticulum membranes. Punctate fluorescence assumes a polar distribution throughout mitosis. Cones of diffuse fluorescence in the chromosomse-to-pole regions of the metaphase spindle appear to coincide with the kinetochore fibers; during anaphase, the cones of fluorescence coalesce and this region of the spindle exhibits uniform diffuse fluorescence. Perturbation of the cellular Ca++ distribution by treatment with lanthanum, procaine, or EGTA results in a loss of diffuse fluorescence with no accompanying change in the intensity of punctate fluorescence. Detergent extraction of cellular membranes causes a total elimination of CTC fluorescence. CTC fluorescence of freshly teased crayfish claw muscle sarcoplasmic reticulum coincides with the A bands and is reduced by perfusion with lanthanum, procaine, and EGTA in a manner similar to that for diffuse fluorescence in the endosperm cells. These results are consistent with the hypothesis that a membrane system in the chromosome-to-pole region of the mitotic apparatus functions in the localized release of sequestered Ca++, thereby regulating the mechanochemical events of mitosis.     

Cellularisation in the endosperm of Arabidopsis thaliana is coupled to mitosis and shares multiple components with cytokinesis

Distinct forms of cytokinesis characterise specific phases of development in plants. In Arabidopsis, as in many other species, the endosperm that nurtures the embryo in the seed initially develops as a syncytium. This syncytial phase ends with simultaneous partitioning of the multinucleate cytoplasm into individual cells, a process referred to as cellularisation. Our in vivo observations show that, as in cytokinesis, cellularisation of the Arabidopsis endosperm is coupled to nuclear division. A genetic analysis reveals that most Arabidopsis mutations affecting cytokinesis in the embryo also impair endosperm cellularisation. These results imply that cellularisation and cytokinesis share multiple components of the same basic machinery. We further report the identification of mutations in a novel gene, SPATZLE, that specifically interfere with cellularisation of the endosperm, but not with cytokinesis in the embryo. The analysis of this mutant might identify a specific checkpoint for the onset of cellularisation.     

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.     

Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.     

Action of taxol on mitosis: modification of microtubule arrangements and function of the mitotic spindle in Haemanthus endosperm

We have studied the effect of taxol on mitosis in Haemanthus endosperm. Immuno-Gold Stain (IGS), a new immunocytochemical method (17), was used to visualize microtubules (MTs) in the light microscope. Observations on MT arrangements were correlated with studies in vivo. Chromosome movements are affected in all stages of mitosis which progresses over at least 10(4) range of taxol concentrations. The three most characteristic effects on MTs are: (a) enhancement of the lateral associations between MTs, seen especially during the reorganization of the polar region of the spindle, (b) promotion of MT assembly, leading to the formation of additional MTs in the spindle and MT arrays in the cytoplasm, and (c) an increase in MT stability, demonstrated in their increased cold resistance. In this report, the emphasis is on the primary, immediate effects, occurring in the first 30 min of taxol action. Effects are detected after a few mins, are reversible, and are concentration/time dependent. The spindle and phragmoplast are remarkably modified due to the enhancement of lateral associations of MTs and the formation of abundant nonkinetochore and polar, asterlike MTs. The equatorial region of the interzone in anaphase may be entirely depleted of MTs, and the spindle may break perpendicular to the spindle axis. Mitosis is completed in these conditions, providing evidence for the motile autonomy of each half-spindle. Trailing chromosome arms in anaphase are often stretched and broken. Chromosome fragments are transported away from the polar regions, i.e., in the direction opposite to that expected (5, 6). This supplies the first direct evidence of pushing by elongating MTs in an anastral higher plant spindle. These observations draw attention to the relation between the lateral association of MT ends to assembly/disassembly and to the role of such an interaction in spindle function and organization.     

Events during the first four rounds of mitosis establish three developmental domains in the syncytial endosperm of Arabidopsis thaliana

Summary. Endosperm begins development as a single fertilized cell that undergoes many rounds of mitosis without cytokinesis resulting in a syncytium. The multinucleate cytoplasm is organized by nucleus-based radial microtubule systems into nuclear-cytoplasmic domains. When microtubules are organized into mitotic spindles, the integrity of the common cytoplasm is maintained by an unaltered network of filamentous actin. The first four rounds of mitosis result in the establishment of three developmental domains within the common cytoplasm. The spindles of the first two rounds of mitosis are oriented parallel to the long axis of the central cell, resulting in four nuclear-cytoplasmic domains in a filamentous arrangement. A switch in spindle orientation occurs in the third round of mitosis; all four spindles are oriented perpendicular to the long axis resulting in eight nuclear-cytoplasmic domains arranged in two adjacent files. Whereas the first three rounds of mitosis are synchronous, the fourth occurs as a wave of successive mitoses that begins at the micropylar pole. By the 16-nuclei stage, differences in nuclear shape, cytoskeletal arrays, and cytoplasmic characteristics mark the differentiation of the syncytium into micropylar, central, and chalazal developmental chambers. Nuclei in the micropylar chamber are fusiform and sheathed by parallel microtubules that flare from their tips, while those in the central and chalazal chambers are spherical. Nuclei in the central chamber are surrounded by radial microtubule systems, while those in the chalaza are enmeshed in a reticulum of microtubules. Whereas the cytoplasm in both micropylar and chalazal chambers is dense and nearly nonvacuolate, the syncytium in the central chamber consists of a single layer of evenly spaced nuclear-cytoplasmic domains surrounding a large central vacuole.Correspondence and reprints: Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, U.S.A.Present address: Department of Plant Sciences, University of Arizona, Tucson, Arizona, U.S.A.     

Birefringence changes in vertebrate striated muscle

The changes in birefringence in the rigor to relax transition of single Triton-extracted rabbit psoas muscle fibers have been investigated. The total birefringence of rigor muscle fibers was dependent on sarcomere length and ranged from (1.46 ± 0.08) × 10⁻³ to (1.60 ± 0.06) ± 10⁻³ at sarcomere lengths from 2.70 μm to 3.40 μm. An increase in total birefringence was measured dependent on sarcomere length when 55 single fibers were relaxed from the rigor state with Mg-ATP. Pyrophosphate relaxation produced a smaller increase in retardation when compared to Mg-ATP. The expected change in intrinsic birefringence during the rigor to relax transition was calculated assuming a hinge function of the subfragment 2 moiety of myosin. The changes in birefringence during isometric contraction and relaxation have been discussed in relation to possible structural changes.     

Birefringence of actin.

The total strain birefringence of F-actin isolated from chicken gizzards was measured as a function of elongation in thin transparent films. Each film held at a certain elongation in a jig was allowed to swell in a penetrating but nondissolving liquid. Seven liquids with different refractive indices were employed. The thickness of the film in each swelling liquid was obtained once equilibrium was established. At each elongation, from 0 to 16%, a Wiener curve was obtained. The minima of the Wiener curves yielded the intrinsic birefringence of F-actin as a function of elongation. The intrinsic birefringence increases with elongation up to 16%, above which the thin films break. The form birefringence at a set refractive index also increases with elongation. The implication of the strain birefringence of F-actin is discussed as it affects the optical properties, mainly light scattering, of tissues such as the fiber cells of lens of the eye.     

Birefringence of tropomyosin crystals.

The birefringence of tropomyosin crystals was measured in the temperature range 5 degrees-35 degrees C. The experimental results are compared with a simple model calculation based on the theory developed by Wiener for the optical properties of colloidal systems. The difference between experimental and theoretical values is less than 15%, which denotes a good agreement given the simplicity of the model. A value of 0.011 was obtained for the intrinsic birefringence of the tropomyosin molecule. The temperature dependence of the crystal birefringence could be accounted for in part by a change of the unit cell parameters; this change was experimentally observed by others in x-ray diffraction experiments.     

Birefringence of the mammalian midbody

The midbody of HeLa cells was studied with polarization and electron microscopy. A band of positive birefringence was found to be associated with the midbody. The characteristic features of this pattern of birefringence indicate that it is not the result of form or intrinsic birefringence, but rather seems to be an example of edge birefringence as described by Inoué [2].     

Starch biosynthesis in cereal endosperm

Stored starch generally consists of two d-glucose homopolymers, the linear polymer amylose and a highly branched glucan amylopectin that connects linear chains. Amylopectin structurally contributes to the crystalline organization of the starch granule in cereals. In the endosperm, amylopectin biosynthesis requires the proper execution of a coordinated series of enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), whereas amylose is synthesized by AGPase and granule-bound starch synthase (GBSS). It is highly possible that plastidial starch phosphorylase (Pho1) plays an important role in the formation of primers for starch biosynthesis in the endosperm. Recent advances in our understanding of the functions of individual enzyme isoforms have provided new insights into how linear polymer chains and branch linkages are synthesized in cereals. In particular, genetic analyses of a suite of mutants have formed the basis of a new model outlining the role of various enzyme isoforms in cereal starch production. In our current review, we summarize the recent research findings related to starch biosynthesis in cereal endosperm, with a particular focus on rice.     

Mitosis in Tilia americana endosperm

The endosperm cells of the American basswood Tilia americana are favorable experimental material for investigating the birefringence of living plant spindles and anaphase movement of chromosomes. The behavior of the chromosomes in anaphase and the formation of the phragmoplast are unique. The numerous (3 n equals 123), small chromosomes move in precise, parallel rows until midanaphase when they bow away from the poles. Such a pattern of anaphase chromosome distribution has been described once before, but was ascribed to fusion of the chromosomes. The bowing of chromosome rows in Tilia is explainable quantitatively by the constant poleward velocity of the chromosomes during anaphase. Peripheral chromosomes are moving both relative to the spindle axis and laterally closer to the axis, whereas chromosomes lying on the spindle axis possess no lateral component in their motion, and thus at uniform velocity progress more rapidly than peripheral chromosomes relative to the spindle axis. The chromosomes are moved poleward initially by pole-to-pole elongation of the spindle, then moved farther apart by shortening of the kinetochore fibers. In contrast to other plant cells where the phragmoplast forms in telophase, the phragmoplast in Tilia endosperm is formed before midanaphase and the cell during midanaphase, while the chromosomes are still in poleward transit.     

Double recombinants in mitosis

A mathematical model for the random process of repeated cell division and recombination in two nonoverlapping genetic intervals is formulated and investigated. From this model, a test for statistical independence of recombination in the two intervals and a method of estimating the rate of double recombination are developed. Crossing over in both intervals, crossing over in one and gene conversion in the other, and gene conversion in both are treated. For markers on the same chromosome, all possible arrangements of the loci relative to the centromere are considered.Supported by National Science Foundation Grant DEB81-03530.