Structural interaction of cytoskeletal components

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament “branches” are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the “microtrabecular network” (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).     

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium

Summary. The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 μM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 μM) only. Correspondence and reprints: Centre of Phycology, Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Třeboň, Czech Republic.     

Organization of cytoskeleton during the tentacle contraction and cytostome movement in the dinoflagellate Noctiluca scintillans McCartney

Summary Electron microscopy of Noctiluca scintillans reveals that the cytoskeleton of the tentacle involved in the motor action of the prey capture consists of three characteristic elements: a deformable peripheral fibrillo-granular ectosarc, abundant underlying microtubules organized in several rows on the convex side, and helicoid filaments about 8 nm in diameter organized into striated myonemes. Microtubules of the external row are crossed-linked with each other by fibrous elements 5 nm in diameter and 10–15 nm long, their links with the second row result in a Y-shaped binding. Bonds of the other rows are linked to each other irregularly between those of the same row. Striated myonemes are regularly inserted between the rows of microtubules on the ectosarc and between its pleats, joining together in a knot of disarrayed filaments with multidirectional orientation in the central axis of the tentacle. Striation of myonemes is based on an alternation of thick striae (TS) 40 nm wide with a periodicity of about 200 nm, and of some intermediary fine striae (FS) 10 nm wide. The events during tentacle contraction are: (1) Rotation of the tentacle, bringing the convex side to the inner side of it. Here, large numbers of microtubules have been visualized by optical immunocytochemistry after labelling with Paramecium antitubulin antiserum. (2) Increase of pleat amplitude (200–300 nm to 600 nm) in concomitance with a decrease of its period (500–700 nm to 250 nm). (3) Apparent modification of the microtubule orientation. (4) Transformation of some TS in several FS without modification of the striation periodicity.Near the cytostome, the cytoskeleton consists of a number quantity of microtubules underlying a non-pleated ectosarc and long tracts of contractile myonemes formed by 6-nm helicoid filaments linking the internal side of the cytostome of the supporting rod. Semirelaxed myonemes show an alternation of fine striae (FS) 35 nm wide between two clear areas (CA) with a periodicity of about 300 nm, plus an incipient dark area (DA) lying between them; together they are transformed into a thick stria (TS) during maximal contraction; the striation periodicity thus decreases by one half. These two systems are compared with one another and with other motile systems.     

The cytoskeleton of cryofixed Purkinje cells of the chicken cerebellum

Summary Cerebella of 3- to 6-week-old chickens were cryofixed in a nitrogen-cooled propane jet, deep-etched and rotary-shadowed. The use of a brief perfusion of 0.32 M sucrose improved the quality of the cryofixation and allowed the study of the deeper layers of the cerebellar cortex. It is reported that the cytoskeleton of the Purkinje cells (PC) shows distinct domains and composition of filamentous structures in the different regions of the cell cytoplasm, such as the perikaryon, the cytoplasm of dendrites and the axoplasm. The perikaryon is occupied by a meshwork of fine filaments, 4–7 nm in diameter, that extends from the nuclear outer membrane to the cell membrane. In this zone the cell organelles (e.g., endoplasmic reticulum, mitochondria) adopt a circular arrangement around the nucleus. All structures are anchored by microfilaments to the cytoplasmic network. The dendrites show a dense cytoplasmic network including bundles of microtubules, neurofilaments and microfilaments. Numerous aggregated globular components are attached to this cytoskeleton. The cytoskeleton of the dendritic spines shows axially oriented 10-nm bundles of filaments, which are interconnected and anchored also to the cell membrane and the components of the agranular endoplasmic reticulum by cross-linkers. As described in peripheral nerves, the axoplasm of axons in the central nervous system exhibits predominantly neurofilaments and microtubules aligned along the axis of the neuntes in a three-dimensional arrangement and interconnected by cross-linker filaments and filamentous structures.     

The cytoskeleton of the fiber cells of Trichoplax adhaerens (Placozoa)

Summary The cytoskeleton of Trichoplax adhaerens fiber cells was studied after chemical fixation, freeze-substitution, lysis of attached cells with nonionic detergents and by immunofluorescence. Cytoskeletal elements present in the cell bodies and reaching into the extensions include microtubules, intermediate filaments, 6–7 nm and 2–3 nm microfilaments. The latter seem to interconnect other cytoskeletal elements. Actin-like microfilaments are found both as networks and parallel strands. Immunofluorescence with antiactin shows the presence of actin in the cell body, underneath the plasmalemma and within the extensions. Both the results of immunofluorescence and the identification of 6–7 nm actin-like microfilaments support the concept of contractility of the fiber cells as the cause of the rapid shape changes of Trichoplax. Anti-tubulin fluorescence corresponds to the location of microtubules in the extensions as well as the cell bodies of the fiber cells. The extensions are withdrawn upon depolymerization of the microtubules by colchicine.     

Assessing the Cytoskeletal System and its Elements in C6 Glioma Cells and Astrocytes by Atomic Force Microscopy

Object To investigate how the characteristic structure of the cytoskeleton in glioma cells is associated with invasiveness. Methods The whole cytoskeletal system was characterized by atomic force microscopy (AFM), while single cytoskeletal elements were exhibited by AFM and using cytoskeletal protein inhibitors to inhibit microfilaments or/and microtubules and displayed by immunofluorescence microscopy. The fluorescence intensity of F-actin was measured by flow cytometry and the structural difference between C6 glioma cells and astrocytes was studied. Results Cytoskeletons in both cells presented network structures, however, the C6 glioma cells showed an irregular edge root and their microfilaments were creber and dense. Intermediate filaments were extensive network structure with non-polarized multipoint connections. The microtubules were relatively big and long and formed tight bundles with close connections between bundles. Astrocytes had a regular and smooth edge, with sparse microfilaments, while the intermediate filaments were dense and interwoven and the microtubules were dense bundled, but only loosely connected each other. Besides, the fluorescence intensity of F-actin was significantly higher in C6 glioma cells (202.54 ± 11.06) than in the astrocytes (62.64 ± 10.23), P < 0.01. Conclusion Whole cytoskeleton and its elements of C6 cells were disclosed of characteristic structures associated with invasiveness. Meanwhile, the content of F-actin could be used as a parameter for measuring cell invasiveness.     

Vimentin Filaments Follow the Preexisting Cytokeratin Network during Epithelial–Mesenchymal Transition of Cultured Neonatal Rat Hepatocytes

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial–mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell–cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colce- mide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell–cell contact and cell shape.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Electron microscopic study of the cytoskeleton of human podocytes

The ultrastructural study of man's cytoskeleton of podocytes is carried out. Populations of podocytes with two different types of structure of the cytoskeleton in dependence on age (2, 4, 6, 37 and 65 years) is revealed in kidneys. The first type of cytoskeleton of the podocyte is peculiar for children's age and is characterized by branched, high density microfilament network, expressed by system of microtubules and single myofilaments. The intermediate filaments here are either utterly absent or present so feebly they find themselves "disguised" by other strongly developed components of cytoskeleton and revealing them with the help of technique of electron microscope is impossible. In kidneys of adults, and especially of old aged persons podocytes with other type of organization of the cytoskeleton are mainly identified. The distinctive signs of the last are bundle arrangement of microfilaments, plural bundles of intermediate filaments and individual microtubules. This study permits to make a conclusion that during individual development and growing old in kidneys of high animals and man, probably, physiological changes causing morphological reconstruction of cytoskeleton which is accompanied by intensive development of intermediate filaments' system with simultaneous "involution" of microtubules and microfilaments' systems take place.     

Polarized Expression of HD1: Relationship with the Cytoskeleton in Cultured Human Colonic Carcinoma Cells

Hemidesmosomes (HDs) mediate adhesion of epithelial cells to the extracellular matrix and have morphological associations with intermediate-size filaments (IFs). Hemidesmosomal molecular components including HD1, the two bullous pemphigoid antigens, and the integrin α6β4 have been identified in HDs of stratified and complex epithelium. In this study, we report that HT29-Fu cells, a human colonic tumor cell line, express two hemidesmosomal components (HD1, α6β4) associated in an adhesion structure termed type II HDs. Immunofluorescence studies showed a colocalization of HD1 and α6β4 in basal patches between actin stress fibers. Using cytochalasin B or vinblastine, two drugs which disrupt the cytoskeleton, we demonstrate that the redistribution of HD1 was probably induced by the reorganization of the basal cytokeratin network. We also show thatin vitroHD1 binds to polymerized cytokeratin intermediate filaments; this suggests that HD1 in intestinal epithelial cells functions as a linker protein connecting cytokeratin filaments to the basal plasma membrane, probably through the β4 subunit of the integrin α6β4.     

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.     

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 μM) or cytochalasin D (10 μM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μM) and prunin (100 μM) as well as KN-62 (10 μM), a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.     

Aggregation of melanosomes in melanophores is accompanied by the reorganization of microtubules and intermediate filaments

The structure of the cytoskeleton in cultured melanophores of the fish Gymnocorymbus ternetzi during aggregation of melanosomes was studied. It has been shown that the motion of pigment granules is accompanied by a reorganization of microtubules and intermediate filament systems. In melanophores with dispersed pigment granules, microtubules are wavy and form a loose network whilst intermediate filaments in such cells form a dense network around the dispersed melanosomes. During aggregation microtubules and intermediate filaments become radially oriented. It was also shown that the surface area of melanophores increased during aggregation.     

Cytoskeleton of the unfertilized sea urchin egg

Unfertilized Paracentrotus lividus egg cytoskeleton is prepared by mild, nonionic detergent extraction at 4 degrees C in buffer systems containing either 2-methyl-2,4-pentanediol (hexylene glycol) or glycerol. These extractions allow the isolation of cytomatrices that maintain the egg form and are 70-80 micron in diameter. DNase inhibition assays show that actin is in polymerized form in these cytomatrices. Ultrastructural observations reveal that the cytoskeletons are made up essentially of 2 categories of filaments, 7-8-nm and 2-4-nm in diameter, respectively. After heavy meromyosin labelling, short, radiating actin filaments are seen in the cortical region, while longer actin filaments are found in the internal region of these cytomatrices. The 2-4-nm filaments of still unknown biochemical nature are organized in a meshwork. In contrast to results found with fertilized eggs, bundles of actin filaments and microtubules are absent; 8-13-nm filaments are not detected.     

Ultrastructural changes during reticulopod withdrawal in the foraminiferan protozoanAllogromia sp., strain NF

Summary Allogromia sp. is a benthic foraminiferan protozoan which extends and withdraws a dynamic network of branching and anastomosing pseudopodia,i.e., reticulopods. Each reticulopod contains an elongate cytoskeleton composed primarily of microtubules (MT). When withdrawal was induced with artificial seawater supplemented with MgCl₂, we found a time-dependent decrease in the number of reticulopodial MTs and a concomitant increase in 5-nm-diameter helical filaments. During the initial stages of withdrawal these helical filaments associated laterally to form loose aggregates. Later they formed dense paracrystalline aggregates, which appeared similar to those seen in the cell bodies of untreatedAllogromia juveniles prior to network extension. Similar results were obtained when withdrawal was induced by using seawater supplemented with other salts (NaCl, KCl). Treatment with an isotonic seawater substitute with an altered Na⁺:K⁺ ratio induced a momentary withdrawal, after which the organism recovered and reextended a network. During the withdrawal phase of this response, MTs became less abundant and aggregates of helical filaments more conspicuous. Together with earlier observations these findings suggest that helical filaments and paracrystalline material are an alternative or intermediate assembly form of MT proteins.     

Human leukocytes as viewed by stereo high-voltage electronmicroscopy

Whole-mount preparations of adherent leukocytes were investigated by stereo high-voltage electronmicroscopy (HVEM) to determine the organization of the cytoplast in unstimulated, motile, and phagocytosing cells. A highly ordered structured cytoplast is revealed. All cytoplasmic organelles are held within an intricate network of fine strands, termed the microtrabecular lattice (MTL), which appears more complex in neutrophils than eosinophils or monocytes. In neutrophils, the tendency of the MTL to expand and contract during cell movement and the responding deformability of the granules appear to influence granule shape. This pleomorphism in granule shape is particularly prominent in exceptionally elongated neutrophils that have not established directionality and demonstrate the appearance of having two leading lamellipodia. Results suggest that the morphology of neutrophil granules is influenced by cell motility, and may account for the pleomorphic populations of granules observed by standard transmission EM. Examination of the cytoskeleton of these elongated cells after detergent extraction reveals separation of the centrosome into two solitary centrioles, with each centriole surrounded by an aster of microtubules. A complex network of microfilaments, intermediate filaments, and microtubules is integrated within a thin area of cytoplasm separating the two cell bodies. Interaction between the MTL, microfilaments, intermediate filaments, and microtubules probably influences granule translocation in these elongated cells. Phagocytosis stimulates a reorganization of the cytoplast; all organelles are found in more central areas of the cytoplasm, bordered by a thin area of hyaloplasm. The MTL appears to limit cytoplasmic granules to a compartment around phagocytic vacuoles, which probably provides the framework for efficient phagolysosome fusion.