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The interaction between the mitotic spindle and the cellular cortex is thought to play a critical role in stimulating cell cleavage. However, little is understood about the nature of such interactions, particularly in tissue culture cells. We have investigated the role of the spindle midzone in signaling cytokinesis by creating a barrier in cultured epithelial cells with a blunted needle, to block signals that may emanate from this region. When the barrier was created during metaphase or early anaphase, cleavage took place only on the sides of the cortex facing the mitotic spindle. Microtubules on the cleaving side showed organization typical of that in normal dividing cells. On the noncleaving side, most microtubules passed from one side of the equator into the other without any apparent organization, and actin filaments failed to organize in the equatorial region. When the barrier was created after the first minute of anaphase, cells showed successful cytokinesis, with normal organization of microtubules and actin filaments on both sides of the barrier. Our study suggests that transient signals from the midzone of early anaphase spindles are required for equatorial contraction in cultured cells and that such signaling may involve the organization of microtubules near the equator.

The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These results demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA.

The mechanism of cytokines is an old problem in cell biology that has received fresh attention recently with a large variety of powerful approaches and experimental systems. Significant advances have been made on the structure of the cortical cytoskeleton, the identification of proteins and genes involved, and the regulatory mechanism. Many surprises have surfaced within the past two years, leading us toward a major revision in our understanding of this important process.

We have used fluorescent latex beads to label membrane receptors on cultured NRK cells. Movement of individual beads during cell division was recorded with digital imaging techniques. Surface-bound beads showed no organized movement during metaphase but started to migrate toward the equator approximately 1 min after anaphase onset, when chromosomes moved out of the equatorial region to create the interzone. The movement was most active in the central region of the cell near separating chromosomes, while beads located near the poles of the cell underwent primarily random motion. Most beads showed a surge in speed upon the passage of chromosomes, suggesting a possible link between chromosome separation and cortical reorganization. Furthermore, treatment of anaphase cells with cytochalasin D induced a rapid, simultaneous collapse of beads and cortical actin filaments into aggregates, indicating that the movement of beads was closely related to the reorganization of the actin cortex. In contrast to normal directional movement, cytochalasin-induced movement occurred in random directions and caused some beads in the equatorial region to move toward poles. Our results indicate that cytokinesis involves contractile activities, not only along the equator, but over a wide area of the actin-containing cortex. In addition, organized cortical activities appear to be temporally activated at anaphase onset, and spatially modulated by the spindle interzone or separating chromosomes.

Publisher Summary This chapter focuses on the needle microinjection and scrape loading of protein molecules into cultured somatic cells. The presence of a fluorescent marker in the injection solution can greatly facilitate microinjection. It allows the examination of flow and easy identification of injected cells. It is critical to obtain an optimal shape of the microneedle by adjusting the temperature and force of the pipette puller. Each needle can be used for more than 50 cells, assuming that the solution for microinjection is reasonably clean. The volume delivered by needle microinjection is usually in the range 1 to 10% cell volume. With some practice, the variability can be maintained to within a factor of 2. A number of factors can affect the volume, including the pressure, tip size, accumulation of proteins at the tip, and presence of any air bubbles in the needle. Compared with needle microinjection, the amount of delivery varies more extensively among scrape-loaded cells. The yield of injected cells and the extent of cell damage also vary among cell types. Cell damage often manifests as the appearance of large vacuoles.

INTRODUCTIONRecent developments in micropatterning techniques have facilitated systematic investigations of how cells respond to geometric cues, which likely have an important role in many physiological processes, such as growth and differentiation. Micropatterned substrates also offer technical advantages for cell biological studies by normalizing cell shapes and reducing the variability among cultured cells. In this article, we present a simple, economical micropatterning method that may be easily implemented in most laboratories. We also describe how to treat the substrate with linear polyacrylamide (PAA) to inhibit unwanted cell adhesion. First, glass coverslip surfaces are activated with 3-methacryloxypropyltrimethoxysilane (bind-silane), an approach that was originally used for covalently linking PAA gels to glass plates for SDS-PAGE to prevent gels from shrinking and swelling during staining/destaining. The activated surfaces are then coated with the photoresist SPR-220, exposed to ultraviolet (UV) light through a photomask, and developed to selectively remove UV-exposed photoresist. Finally, linear PAA is grafted onto the exposed areas of the bind-silane substrate (to prevent cell adhesion), and the remaining photoresist is removed to uncover the glass surface, making the unexposed areas available as adhesive areas for cell culture.

In the title compound, C13H13Cl2N3OS, the thiazole and benzene rings are roughly parallel to one another in two layers [dihedral angle = 5.08 (2)°] because the N—C—C—N—C chain that links the two rings is folded [N—C—C—N torsion angle = 12.0 (2)°] rather than fully extended. An intramolecular N—H...N interaction occurs. In the crystal, weak intermolecular N—H...N and C—H...O interactions are present and π–π interactions are indicated by the short distances [3.507 (3)–3.665 (2) Å] between the centroids of the thiazole and benzene rings.

Thymosin beta 4 (beta 4) is a 5-kDa polypeptide originally identified in calf thymus. Although numerous activities have been attributed to beta 4, its physiological role remains elusive. Recently, beta 4 was found to bind actin in human platelet extracts and to inhibit actin polymerization in vitro, raising the possibility that it may be a physiological regulator of actin assembly. To examine this potential function, we have increased the cellular beta 4 concentration by microinjecting synthetic beta 4 into living epithelial cells and fibroblasts. The injection induced a diminution of stress fibers and a dose-dependent depolymerization of actin filaments as indicated by quantitative image analysis of phalloidin binding. Our results show that beta 4 is a potent regulator of actin assembly in living cells.

Recent studies indicate that extracellular mechanical properties, including rigidity, profoundly affect cellular morphology, growth, migration, and differentiation [R. J. Pelham, Jr. and Y. Wang, Proc. Natl. Acad. Sci. U. S. A., 1997, 94(25), 13661–13665; H. B. Wang, M. Dembo and Y. L. Wang, Am. J. Physiol. Cell Physiol., 2000, 279(5), C1345–C1350; P. C. Georges, and P. A. Janmey, J. Appl. Physiol., 2005, 98(4), 1547–1553; C. M. Lo, H. B. Wang, M. Dembo and Y. L. Wang, Biophys. J., 2000. 79(1), 144–152; D. E. Discher, P. Janmey and Y. L. Wang, Science, 2005, 310(5751), 1139–1143; A. J. Engler, M. A. Griffin, S. Sen, C. G. Bonnemann, H. L. Sweeney and D. E. Discher, J. Cell Biol., 2004, 166(6), 877–887]. However, most studies involving rigidity sensing have been performed by comparing cells on separate substrata of fixed stiffness. To allow spatial and/or temporal manipulation of mechanical properties, we have developed a modulatable hydrogel by reacting linear polyacrylamide (PA) with a photosensitive crosslinker. This material allows UV-mediated control of rigidity, softening by 20–30% upon irradiation at a dose tolerated by live cells. Global UV irradiation induces an immediate recoiling of 3T3 fibroblasts and a reduced spread area at steady state. Furthermore, localized softening of the posterior substratum of polarized cells causes no apparent effect, while softening of the anterior substratum elicits pronounced retraction, indicating that rigidity sensing is localized to the frontal region. This type of material allows precise spatial and temporal control of mechanical signals for both basic research and regenerative medicine.

Although actin filaments are known to be localized in the cortex of axons and in the growth cones of nerve cells, it is unclear how actin-containing structures are assembled during nerve growth. We have studied the formation of actin structures in growing neurites by microinjecting fluorescent phalloidin or actin into PC12 neuron-like cells to label endogenous actin filaments. Upon stimulation of neurite growth in cells microinjected with fluorescent phalloidin, little or no fluorescence was detected in nascent growth cones and adjacent neurites despite the presence of actin filaments in these regions, suggesting that actin filaments were primarily formed by de novo assembly rather than the transport and reorganization of pre-existing, phalloidin-labeled actin filaments. Time-lapse observations of the distribution of phalloidin-labeled actin filaments during neurite elongation confirmed that fluorescence associated with pre-existing neurite cortex spread out more slowly than the elongation of neurites. Furthermore, when a dark spot was photobleached with a laser microbeam along neurites of cells microinjected with either fluorescent phalloidin or actin, the spot showed no appreciable translocation during active neurite elongation. Taken together, these results suggest that de novo assembly of actin filaments plays a crucial role in the formation of growth cones and adjacent cortex in the distal region of neurites, but does not appear to require the anterograde or retrograde transport of cortical filaments, or the passive stretching of the proximal segment of the neurite cortex.

Myosin light chain kinase (MLCK) is thought to regulate the contractile activity in smooth and non-muscle cells, and may play an important role in controlling the reorganization of the actin-myosin cytoskeleton during cell division. To test this hypothesis we have microinjected the 61-kD catalytic fragment of MLCK into mitotic cells, and examined the effects of unregulated MLCK activity on cell division. The microinjection of active 61 kD causes both a significant delay in the transit time from nuclear envelope breakdown to anaphase onset, and an increase in motile surface activity during and after metaphase. Control experiments with intact MLCK or with inactive catalytic fragment suggest that these effects are specifically induced by the unregulated myosin light chain kinase activity. Immunofluorescence analysis suggests that delays in mitosis are coupled to disruptions of spindle structures, while increased surface motility may be related to changes in the organization of actin and myosin at the cell cortex. Most importantly, despite the expression of strong phenotypes, 61 kD-injected cells still form functional cleavage furrows that progress through cytokinesis at rates identical to those of control cells. Together, these results suggest that the activity of MLCK can affect mitosis and cortical activities, however additional control mechanisms are likely involved in the regulation of cytokinesis.

We have studied the nuclear localization of rhodamine-labeled pre-mRNA after microinjection into nuclei of cultured rat kidney epithelial cells. Intranuclear localization of the injected RNA was followed in the living cells by fluorescence microscopy and digital image processing. Injected human beta-globin pre-mRNA became localized in 30-60 discrete nuclear sites that were coincident with loci defined by monoclonal antibodies against small nuclear ribonucleoproteins (Sm) or another spliceosome component (SC-35) in parallel immunocytochemical studies on the same nuclei. Similar patterns of nuclear localization were observed with a rat proenkephalin pre-mRNA. Nuclear microinjection of an intronlacking beta-globin RNA, a splicing-defective beta-globin mutant pre-mRNA, or an antisense beta-globin pre-mRNA did not result in localization at discrete sites. These results indicate that pre-mRNA binds preferentially to nuclear Sm and SC-35 antibody-reactive sites in vivo and that the binding requires intron sequences.

Publisher Summary This chapter describes a method for the fluorescent labeling of the myosin sulfhydryl groups with tetramethylrhodamine, a probe with optimal fluorescence properties for applications in living cells. The method of labeling applies to both skeletal and smooth muscle myosins, although the discussion will focus on the smooth muscle myosin. The method uses tetramethylrhodamine iodoacetamide (TRIA) as the reagent for attaching the tetramethylrhodamine fluorlophore to the sulfhydryl groups of the myosin molecule. The procedure is similar to that for other iodoacetamides as summarized by Reisler. However, special steps are taken to overcome the problems of the relatively low solubility of TRIA and the difficulty in removing unreacted TRIA from proteins. All the buffers should be prepared at 4° and all steps are performed at 4°. The labeled myosin should contain no detectable free dye molecules. The extent of labeling is measured by standard methods: the protein concentration is determined by the Lowry assay, the concentration of tetramethylrhodamine is determined by measuring the absorbance at 555 nm. The molar concentrations of the protein and the fluorophore are then calculated based on the molecular weight of myosin (470,000) and the extinction coefficient of bound tetramethylrhodamine 5 (47,000 M -1 cm -1 ). The labeling protocol yields typically a conjugate with four fluorophores per smooth muscle myosin molecule.

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.

Most nonmuscle cells are known to maintain a relatively high concentration of unpolymerized actin. To determine how the polymerization of actin is regulated, exogenous nucleation sites, prepared by sonicating fluorescein phalloidin-labeled actin filaments, were microinjected into living Swiss 3T3 and NRK cells. The nucleation sites remained as a cluster for over an hour after microinjection, and caused no detectable change in the phase morphology of the cell. As determined by immunofluorescence specific for endogenous actin and by staining cells with rhodamine phalloidin, the microinjection induced neither an extensive polymerization of endogenous actin off the nucleation sites, nor changes in the distribution of actin filaments. In addition, the extent of actin polymerization, as estimated by integrating the fluorescence intensities of bound rhodamine phalloidin, did not appear to be affected. To determine whether the nucleation sites remained active after microinjection, cells were first injected with nucleation sites and, following a 20-min incubation, microinjected with monomeric rhodamine-labeled actin. The rhodamine-labeled actin became extensively associated with the nucleation sites, suggesting that at least some of the nucleation activity was maintained, and that the endogenous actin behaved in a different manner from the exogenous actin subunits. Similarly, when cells containing nucleation sites were extracted and incubated with rhodamine-labeled actin, the rhodamine-labeled actin became associated with the nucleation sites in a cytochalasin-sensitive manner. These observations suggest that capping and inhibition of nucleation cannot account for the regulation of actin polymerization in living cells. However, the sequestration of monomers probably plays a crucial role.

A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle. We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules. We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle.

Over the past ten years various technical advances have allowed the direct study of molecular activities in cultured cells under a fluorescence microscope. Fluorescent probes are well known for their high sensitivity, specificity and amenability to various spectroscopic analyses. When used in conjunction with low-light-level detectors and image processing computers, high resolution images of weak signals from single cells can be successfully acquired. In addition, the availability of digitized images has greatly facilitated the extraction of photometric and morphometric information.We use Zeiss inverted microscopes equipped with epi-illuminators and Dage-MTI ISIT video cameras or Photometrics cooled CCD cameras. Custom incubator systems built on the microscope stages allow the maintenance of live cells for up to several days. The signals are processed with image processing systems (Imaging Technologies) interfaced with graphics workstations (Silicon Graphics, Model 3130 or 4D/20) or personal computers (386/33). All images are acquired by frame averaging/signal integration, followed by subtraction of the dark noise, and storage as computer files. A variation of this simple processing strategy has allowed the detection of extremely weak signals that are essentially invisible on unprocessed ISIT images. Computer programs are then used to display sequences or images as motion pictures, to measure the linear dimension and angular orientation, and to integrate intensities over defined areas.

We have employed fluorescent analogue cytochemistry and fluorescence photobleaching to study the mobility of actin and alpha-actin along stress fibers. Rhodamine-labeled actin or alpha-actinin microinjected into embryonic chick cardiac fibroblasts soon became incorporated into stress fibers. A pulse of a laser microbeam was used to photobleach small spots on the fluorescent stress fibers. Images of the bleached fiber were recorded with an intensified image processing system at 2-3 min intervals. The distance between the bleached spot and the terminus of the stress fiber, which remained stationary throughout the experiment, was then measured in the successive images. Movement of bleached spots was detected along stress fibers located in the apparently trailing processes of polygonal fibroblasts, and only occurred in one direction: away from the distal tip of the stress fiber. The rate of movement calculated for alpha-actinin-injected cells was 0.24 +/- 0.12 micron/min, for actin-injected cells, 0.29 +/- 0.11 micron/min. The rate did not seem to be affected by the location of the spot relative to the distal end of the stress fiber unless the spot was located within the most distal 5 microns of the stress fiber. Anti-myosin antibody staining indicated that stress fibers which demonstrated translocation were relatively depleted of myosin. The apparent translocation of proteins along stress fibers, possibly generated by stretching, may be related to the retraction of cell processes during locomotion.

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were microinjected with fluorescently labeled alpha-actinin and/or vinculin and observed using video microscopy before and after the addition of 50 micrograms/ml GRGDS. As soon as 5 min after treatment, fluorescent alpha-actinin and vinculin became dissociated simultaneously from the sites of many focal contacts. The proteins either moved away as discrete structures or dispersed from adhesion plaques. As a result, the enrichment of alpha-actinin and vinculin at these focal contacts was no longer detected. The focal contacts then faded away slowly without showing detectable movement. These data suggest that the binding state of integrin has a transmembrane effect on the distribution of cytoskeletal components. The dissociation of alpha-actinin and vinculin from adhesion plaques may in turn weaken the contacts and result in rounding and detachment of cells.

We have combined fluorescent analogue cytochemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3 of that observed in aqueous solution; 90% of the actin has a mobility 1/2 to 1/8 of that of G-actin in aqueous solution, and approximately equal to 10% of the actin has a mobility comparable to that of F-actin in aqueous solution. Therefore, no more than 10% of the actin in the cytoplasm of amoebae can exist as static filaments. Microinjection of phalloidin decreases the diffusion coefficient of the mobile component of cytoplasmic actin, and it also increases the low-mobility fraction to 50% but has no effect on the mobility of labeled ovalbumin. By comparing the mobility of actin in different parts of amoebae and by separating cytoplasm from plasmalemma-ectoplasm, we found the low-mobility fraction of actin to be enriched in the tail, along the plasmalemma-ectoplasm, and in contracted cytoplasm.

A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds 125I-heavy meromyosin and 125I-tropomyosin. The associations between the F-actin affinity matrix and the iodinated F-actin binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

We investigated how actin bundles assemble, disassemble, and reorganize during cell movement. Living chick embryonic fibroblasts were microinjected with actin molecules that had been fluorescently labeled with tetramethylrhodamine. We found that the fluorescent analogue of actin can be used successfully by both existing and newly formed cellular structures. Using time-lapse photography coupled to image-intensified fluorescence microscopy, we were able to detect various patterns of reorganization in motile cells. Assembly of stress fibers occurred near both the leading and the trailing ends of the cell. The initial structure appeared as discrete spots that subsequently extended into stress fibers. The extension occurred unidirectionally. The site of initiation near the leading edge remained stationary relative to the substrate during subsequent cell advancement. However, the orientation of the fiber could change according to the direction of cell movement. In addition, existing stress fibers could merge or fragment. The shortening of stress fibers can occur from either end of the fiber. Shortening from the proximal end (centrifugal shortening) was accompanied by a decrease in fluorescence intensity, as if the bundle were disassembling, and usually led to the total disappearance of the bundle. Shortening from the distal end (centripetal shortening), on the other hand, is usually accompanied by an increase in fluorescence intensity at the distal end of the bundle, as if this end had pulled loose from its attachment and retracted toward the center of the cell. Besides stress fibers, arc-like actin bundles have also been detected in spreading cells. These observations can explain how the organization of actin bundles coordinates with cell movement, and how stress fibers reach a highly regular pattern in static cells.

It has previously been inferred that the fusion of a macrophage secondary lysosome with a phagosome delivers the entire lysosomal contents uniformly to the phagosome. We found, however, that different fluorescent lysosomal probes can enter phagosomes at remarkably different rates, even though they are initially sequestered together in the same organelles. Thus, sulforhodamine is almost exclusively delivered to yeast-containing phagosomes within 2 h of phagocytosis. But fluoresceinated, high molecular weight dextran accumulates in the same phagosomes only over a period of approximately 24 h. We postulate that the delivery of lysosomal contents may involve an intermittent and incremental process in which individual components can be selectively and sequentially transferred.

The technique of molecular cytochemistry has been used to follow the distribution of fluorescently labeled actin in living Chaos carolinensis and Amoeba proteus during ameboid movement and various cellular processes. The distribution of 5-iodoacetamidofluorescein-labeled actin was compared with that of Lissamine rhodamine B sulfonyl chloride-labeled ovalbumin microinjected into the same cell and recorded with an image intensification microscope system. Actively motile cells demonstrated a rather uniform distribution of actin throughout most of the cytoplasm, except in the tail ectoplasm and in plasma gel sheets, where distinct actin structures were observed. In addition, actin-containing structures were induced in the cortex during wound healing, concanavalin A capping, pinocytosis, and contractions elicited by phalloidin injections. The formation of distinct fluorescent actin structures has been correlated with contractile activities.

Actin labeled with 5-iodoacetamidofluorescein has been incorporated into the functional pool of actin in Chaos carolinensis and Physarum polycephalum by direct microinjection. The functional activity of the labeled actin has been analyzed at three levels of organization as: (a) with the purified actin, (b) in motile extracts of cells, and (c) in living motile cells. The labeled actin exhibited normal polymerization and activated myosin ATPase to a similar extent as unlabeled controls. Labeled actin and endogenous actin were incorporated into contracted pellets to approximately the same extent in motile cell extracts. After labeled actin had been microinjected into single C. carolinensis cells, the fluorescent actin spread into both the endoplasm and etoplasm without forming distinct fibrils. In contrast, fluorescent bundles developed in the ectoplasm of P. polycephalum following microinjection of labeled actin. This experimental method in conjunction with fluorescence spectroscopic techniques could become a powerful tool for studying the intracellular distribution and structural changes of components in living cells.

We have used fluorescence analogue cytochemistry in conjunction with time lapse recording to study the dynamics of alpha-actinin, a major component of the Z line, during myofibrillogenesis. Rhodamine-labeled alpha-actinin microinjected into living cultured chick skeletal myotubes became localized in discrete cellular structures within 1 h and remained specifically associated with structures for up to 4 d, allowing individual identified structures to be followed during development. In the most immature cells used, alpha-actinin was found in diffuse aggregates, some of which displayed sarcomeric periodicity. Aggregates were observed to coalesce into better defined structures (Z bands) that were approximately 1.0-micron wide. Z bands condensed into narrow, more intensely fluorescent Z lines in 4-48 h. During this period, Z lines grew laterally, primarily by the addition of small beads of alpha-actinin to existing Z lines or by the merging of small Z lines. In more mature cells, alpha-actinin added to Z lines without going through a visible intermediary structure. Mean sarcomere length did not change significantly during the stages examined, although the variability of sarcomere length did decrease markedly over time for identified sets of sarcomeres. At early stages, myofibrils frequently shifted position in both the longitudinal and lateral directions. Neighboring myofibrils were frequently associated for one or more sarcomeres sporadically along their length, such that the intervening sarcomeres were often misaligned. Associations between myofibrils were often transitory. Shifts in myofibril location in conjunction with the formation, breaking, and reformation of lateral associations between myofibrils facilitated the alignment of Z lines through a trial and error process.