Biomedical subjects

W Gawlitta

Publications and source records attributed to W Gawlitta.

8 recordsLinked to original sources

PubMed1982

Spatial organization and fine structure of the cortical filament layer in normal locomoting Amoeba proteus.

The fine structural organization of a cortical filament layer in normal locomoting Amoeba proteus was demonstrated using improved fixation and embedding techniques. Best results were obtained after application of PIPES-buffered glutaraldehyde in connection with substances known to prevent the depolymerization of F-actin, followed by careful dehydration and freeze-substitution. The filament layer is continuous along the entire surface; it exhibits a varying thickness depending on the cell polarity, measuring several nm in advancing regions and 0.5-1 micron in retracting ones. Two different types of filaments are responsible for the construction of the layer: randomly distributed thin (actin) filaments forming an unordered meshwork beneath the plasma membrane, and thick (myosin) filaments mostly restricted to the uroid region in close association with F-actin. The cortical filament layer generates the motive force for amoeboid movement by contraction at posterior cell regions and induces a pressure flow that continues between the uroid with a high hydrostatic pressure and advancing pseudopodia with low one. The local destabilization of the cell surface as a precondition for the formation of pseudopodia is enabled by the detachment of the cortical filament layer from the plasma membrane. This results in morphological changes by the active separation of peripheral hyaloplasmic and central granuloplasmic regions.

Amoeba↗

PubMed1981

Visualization of actin polymerization and depolymerization cycles during polyamine-induced cytokinesis in living Amoeba proteus.

Microinjection of spermine induces cytokinesis of Amoeba proteus. Within 30--60 s after spermine injection cells form one, or less commonly, two cleavage furrows and within the following 4--10 min the constrictions are completed. The resulting nucleated cell parts show normal streaming and locomotion, whereas the non-nucleated cell parts remain stationary and later degenerate. The intracellular distribution of fully polymerization-competent fluorescently labelled muscle actin was followed by image intensification. Double injection experiments initially using labelled action and 30 min later spermine revealed a ring-like structure of enhanced fluorescence corresponding to the constricting cleavage furrow. Immediately after cleavage was completed, the ring disappeared. Electron microscopy of cells fixed during spermine-induced cytokinesis showed numerous well aligned actin and myosin filaments in the developing cleavage furrow. These filaments are a specialized manifestation of the cell cortex. The results demonstrate that cycles of actin and myosin polymerization and depolymerization and the parallel alignment of preexisting filaments (crosslinking) represent a basic mechanism in the generation of the motive force during cytokinesis.

Actins↗

PubMed1981

Coiling of intermediate filaments induced by microinjection of a vimentin-specific antibody does not interfere with locomotion and mitosis.

Microinjection of polyclonal sheep anti-vimentin IgGs purified by affinity chromatography into a rat fibroblastoid line leads to a specific reorganization of the cytoskeleton. Immunofluorescence microscopy shows that cytoplasmic microtubules and microfilaments are unaffected by intermediate filaments collapse and are collected into a tight perinuclear cap containing antibody-crosslinked vimentin filaments. The crosslinking was further documented by electron microscopy after treatment with Triton X-100 and ferritin-labelled anti-sheep IgGs. Inspite of the presence of the caps, which are retained for about 30 h, cells show a normal morphology and are locomotive. The collapsed intermediate filaments do not interfere with subsequent mitosis or with cytokinesis. After mitosis the capped filaments can be distributed either to both daughter cells or to only one of the two daughter cells.

Animals↗

PubMed1980

Studies on microplasmodia of Physarum polycephalum. I. Classification and locomotion behavior.

Depending on the conditions of the axenic shuttle culture, microplasmodia of the acellular slime mold Physarum polycephalum can be subdivided into three classes regarding fine structural organization and protoplasmic streaming activity: (1) spherical and rod-shaped types, (b) ameboid types, and (c) symmetrical types. In ameboid microplasmodia, the motive force for the irregular protoplasmic streaming activity is generated by alternative contraction and relaxation of a membrane-associated layer, morphologically consisting exclusively of thin filaments (probably actin). The protoplasm flows along a hydraulic pressure gradient produced by the filament layer within limited regions of the cell periphery. In dumbbell-shaped microplasmodia the motive force for the regular protoplasmic shuttle streaming between the two spherical heads is generated both by volume changes of the peripheral cell region (caused by the contractile activity of the membrane-associated filament layer), and by volume changes of the internal cell membrane invagination system (caused by fibrils attached to the basal region of the invaginations). The development from the unordered protoplasmic streaming pattern and less complicated fine structural organization in ameboid microplasmodia to the highly organized protoplasmic shuttle streaming and the more complicated morphology in dumbbell-shaped microplasmodia can be explained by intermediate stages. Whereas the motive force for the transport of smaller amounts of protoplasm can be generated by the exclusive action of a cortical filament layer, the existence of a filament cortex, the display of cytoplasmic fibrils, and the development of plasma membrane invaginations appear to be a necessary precondition for the transport of large amounts of protoplasm.

Cytoplasm↗

PubMed1980

Organization and spatial arrangement of fluorescein-labeled native actin microinjected into normal locomoting and experimentally influenced Amoeba proteus.

Fully polymerization-competent fluorescein-labeled actin from skeletal muscle was microinjected into both normal moving and experimentally treated Amoeba proteus. Its intracellular distribution was followed by integral image intensification of the fluorescence on a television screen and compared with controls injected with rhodamine-labeled serum albumin. The labeled actin was incorporated into the endogenous actin pool and exhibited a characteristic redistribution depending on the cellular morphology. Increased amounts of labeled actin could be detected within a thin layer separating the hyalo- and granuloplasm or running immediately beneath the plasma membrane when hyaloplasmic regions were absent. The topography of the fluorescent layer demonstrated in living cells is in agreement with the cortical microfilament layer described ultrastructurally recently in corresponding cells. The combined results emphasize the important role of the cortical filament layer in both morphogenetic processes (e.g., hyalo-granuloplasm separation or changes in cell shape) and motive force generation for cytoplasmic streaming and amoeboid movement.

Actins↗

PubMed1980

Pinocytosis and locomotion of amoebae. XV. Visualization of Ca++-dynamics by chlorotetracycline (CTC) fluorescence during induced pinocytosis in living Amoeba proteus.

The dynamics of Ca++ during induced pinocytosis were studied in Amoeba proteus using chlorotetracycline (CTC). The fluorescence of the Ca++ - CTC-complex was monitored by an image intensification system, which has certain advantages over standard equipment: (1) Living cells are not subjected to the damaging influence of intensive microscopic illumination, (2) fluorescent probes are not bleached during observation, and (3) the rapid dynamics of the Ca++ -fluxes can be recorded using short exposure times. The results demonstrate the existence of Ca++ bound to intracellular and extracellular sites of the cell membrane complex in normal locomoting and pinocytotic Amoeba proteus. The application of cations inducing pinocytosis causes a rapid decrease in the external CTC-fluorescence probably due to a release of Ca++ from the mucous layer. The degree of fluorescence intensity is correlated with the capacity of pinocytotic channel formation, i.e., the fluorescence decreases as the number of channels increases. During the phase of vesiculation a distinct fluorescence mainly restricted to the basal region of the channels is observed. Intracellular Ca++ was detected in close vicinity to the plasma membrane after both microinjection and external application of CTC. The internal CTC-fluorescence is slightly decreased during the induction phase of pinocytosis. The observations are in good agreement with previous results on the localization of Ca++ -binding sites at the plasma membrane of Amoeba proteus and demonstrate the important role of Ca++ -fluxes for the process of pinocytosis.

Amoeba↗

PubMed1980

The influence of an actin-modulating protein (AM-protein) from Physarum polycephalum on the cell motility of Amoeba proteus.

An actin-modulating protein (AM-protein) isolated from the acellular slime mold Physarum polycephalum and microinjected into living Amoeba proteus causes characteristic changes in cell shape, locomotory behaviour, and organization of the microfilament system of the amebae. The peptide chain weight of the AM-protein, which binds to one actin molecule with high affinity thus forming a heterodimer, is 43 000. The heterodimer is a powerful inhibitor of actin polymerization, when added to G-actin. A 50% inhibition is already obtained at a weight ratio of 0.05 AM-protein/actin. The AM-protein heterodimer has only little effect on F-actin, which can be neglected under the experimental conditions used. The microinjected AM-protein transforms normal locomoting polytactic amebae into orthotactic cell models. The orthotactic forms are characterized by a polarized organization, i.e. a flattened advancing cell pole consisting of one large pseudopod showing clear suppression of further pseudopod formation, and a contracting uroid undergoing cellular budding processes by the formation and vesiculation of numerous small evaginations. The observed changes in morphology can be interpreted as a relaxation of the advancing cell region due to a significant reduction of the polymerizable actin pool. As the AM-protein heterodimer does not attack F-actin filaments or the interaction of actin and myosin the middle region and the uroid of the microinjected cells are able to perform contractions which result in an orthotactic moving pattern. In conjunction with control experiments using double injection of AM-protein and phalloidin our observations prove the important role of controlled actin polymerization and depolymerization processes for cytoplasmic streaming activity and cell locomotion in ameboid cells.

Amoeba↗

PubMed1979

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus. An attempt to explain amoeboid movement.

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.

Amoeba↗