The Fine Structure of Trypanosoma congolense in Its Bloodstream Phase

SYNOPSIS. The fine structure of 2 isolates of Trypanosoma congolense maintained in laboratory rodents has been studied from thin sections of osmium- and aldehyde-fixed flagellates. The pellicular complex, nucleus, and flagellar apparatus are all similar to those of other African trypanosomes. Aberrant intracellular differentiation of the flagellum is occasionally found. As in bloodstream forms of other salivarian trypanosomes the single mitochondrion forms an irregular canal running from one end of the body to the other, with a shallow bowl-shaped expansion forming a capsule for the fibrous kinetoplast (mitochondrial DNA). A connexion between the mitochondrial envelope of the kinetoplast and the basal body of the flagellum is not evident, and sometimes the flagellum base is not even apposed to the kinetoplast but lies behind it. Tubular cristae are present in the mitochondrial canal and, by light microscopy, this structure gives a positive reaction for NAD diaphorase suggesting at least some activity in electron transport, even tho at this stage in its life cycle respiration is doubtfully sensitive to cyanide and cytochrome pigments are in all probability absent. The region of the cytoplasm between the nucleus and the flagellar pocket has all the trappings associated with secretory cells in higher animals, or with the secretion of surface structures in phytoflagellates. just behind the nucleus a limb of granular reticulum subtends a Colgi stack of flattened saccules with attendant vesicles. Close to the distal pole of the Golgi complex is a network of smooth-membraned cisternae, termed here the agranular or secretory reticulum, which undergoes localized swelling with the accumulation of a secretory product to form large spherical sacs or vacuoles. These network-linked vacuoles probably correspond to the post nuclear vacuole complex visible by light microscopy. From its apparent secretory function this complex is regarded here as being possibly an extension or derivative of the Golgi complex, the smooth-membraned tubules lying alongside the 2 structures possibly representing a link between them. By analogy with phytoflagellates and the secretory cells of higher animals, it is suggested that the secretion is transported for discharge into the flagellar pocket by way of multivesicular bodies and smooth-walled tubules or vesicles. Spiny pits in the wall of the flagellar pocket, and similar-sized vesicles in the nearby cytoplasm, could be stages in either exocytosis of secretion or endocytosis (pinocytosis). It is tentatively suggested that the secretion may be the material from which the surface coat is formed. Neither a cytostome nor a contractile vacuole has been observed in T. congolense.     

Differentiation Within the Bacterial Flagellum and Isolation of the Proximal Hook

Purified and crude flagellar isolates from cells of Bacillus pumilus NRS 236 were treated with acid, alcohol, acid-alcohol, or heat, and were examined electron microscopically in negatively stained and shadow-cast preparations. Under certain conditions, each of these agents causes the flagella to break between the proximal hooks and the spiral filaments. In such preparations, filaments are seen in various stages of disintegration, whereas hooks of fairly constant length retain their integrity and morphological identity. When crude isolates of flagella are treated under these conditions, the hooks remain attached to membrane fragments or bear basal material. These findings substantiate previous structural observations that led to the view that the proximal hook is a distinct part of the bacterial flagellum and further confirm that the hook is tightly associated with basal material and the cytoplasmic membrane. It appears that the hook is a polarly oriented structure, and that the interactions between the hook and the basal material or the cytoplasmic membrane are different from those between the hook and the filamentous portion of the organelle. Moreover, both types of interaction apparently differ still from those by which the flagellin subunits are held together in the flagellar filament. Hooks were isolated by exploiting the differences in relative stability shown by the various morphological regions of the bacterial flagellum.     

Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.     

Structural polymorphism in bacterial EspA filaments revealed by cryo-EM and an improved approach to helical reconstruction

The traditional Fourier-Bessel approach to three-dimensional reconstruction from electron microscopic (EM) images of helical polymers involves averaging over filaments, assuming a homogeneous structure and symmetry. We have used a real-space reconstruction approach to study the EspA filaments formed by enteropathogenic E. coli. In negative stain, the symmetry of these filaments is ambiguous, and we suggest that such ambiguities may be more prevalent than realized. Using cryo-EM of frozen-hydrated filaments, we find that these filaments have a fixed twist with 5.6 subunits per turn but an axial rise per subunit that varies from about 3.6 A to 5.6 A. Reconstructions at approximately 15 A resolution show a switching between the more compressed and extended filaments in the packing of putative alpha helices around the hollow lumen. Outside of a crystal, where there is nothing to maintain long-range order, the structural polymorphism in helical polymers may be much greater than has been assumed.     

Determinants of GPI-PLC Localisation to the Flagellum and Access to GPI-Anchored Substrates in Trypanosomes

In Trypanosoma brucei, glycosylphosphatidylinositol phospholipase C (GPI-PLC) is a virulence factor that releases variant surface glycoprotein (VSG) from dying cells. In live cells, GPI-PLC is localised to the plasma membrane where it is concentrated on the flagellar membrane, so activity or access must be tightly regulated as very little VSG is shed. Little is known about regulation except that acylation within a short internal motif containing three cysteines is necessary for GPI-PLC to access VSG in dying cells. Here, GPI-PLC mutants have been analysed both for subcellular localisation and for the ability to release VSG from dying cells. Two sequence determinants necessary for concentration on the flagellar membrane were identified. First, all three cysteines are required for full concentration on the flagellar membrane. Mutants with two cysteines localise predominantly to the plasma membrane but lose some of their flagellar concentration, while mutants with one cysteine are mainly localised to membranes between the nucleus and flagellar pocket. Second, a proline residue close to the C-terminus, and distant from the acylated cysteines, is necessary for concentration on the flagellar membrane. The localisation of GPI-PLC to the plasma but not flagellar membrane is necessary for access to the VSG in dying cells. Cellular structures necessary for concentration on the flagellar membrane were identified by depletion of components. Disruption of the flagellar pocket collar caused loss of concentration whereas detachment of the flagellum from the cell body after disruption of the flagellar attachment zone did not. Thus, targeting to the flagellar membrane requires: a titratable level of acylation, a motif including a proline, and a functional flagellar pocket. These results provide an insight into how the segregation of flagellar membrane proteins from those present in the flagellar pocket and cell body membranes is achieved.     

Role of divalent cations in the subunit associations of complex flagella from Rhizobium meliloti.

Rhizobium meliloti, a symbiotic, nitrogen-fixing soil bacterium with complex flagella, as well as other members of the family Rhizobiaceae, rapidly lost motility when suspended in buffers lacking divalent cations but retained good motility in buffers containing calcium, magnesium, barium, or strontium. Loss of motility was associated with loss of flagella from the cells. Analysis of flagella by sedimentation, gel electrophoresis, and electron microscopy revealed that removal of divalent cations from the complex flagella of R. meliloti resulted in extensive dissociation of the flagellar filaments into low-molecular-weight subunits. Accordingly, divalent cations such as calcium and magnesium that are normally present at high concentrations in the soil solution may be crucial to the assembly and rigidity of complex flagella.     

Plain and Complex Flagella of Pseudomonas rhodos: Analysis of Fine Structure and Composition

Cells of Pseudomonas rhodos 9-6 produce two morphologically distinct flagella termed plain and complex, respectively. Fine structure analyses by electron microscopy and optical diffraction showed that plain flagellar filaments are cylinders of 13-nm diameter composed of globular subunits like normal bacterial flagella. The structure comprises nine large-scale helical rows of subunits intersecting four small-scale helices of pitch angle 25 degrees . Complex filaments have a conspicuous helical sheath, 18-nm wide, of three close-fitting helical bands, each about 4.7-nm wide, separated by axial intervals, 4.7 nm wide, running at an angle of 27 degrees . The internal core has similar but not identical substructure to plain filaments. Unlike plain flagella, the complex species is fragile and does not aggregate in bundles. Mutants bearing only one of two types of flagellum were isolated. Cells with plain flagella showed normal translational motion, and cells with complex flagella showed rapid spinning. Isolated plain flagella consist of a 37,000-dalton subunit separable into two isoproteins. Complex filaments consist of a 55,000-dalton protein; a second 43,000-dalton protein was assigned to complex flagellar hooks. The results indicate that plain and complex flagella are entirely different in structure and composition and that the complex type represents a novel flagellar species. Its possible mode of action is discussed.     

Flagellar systems in the euglenoid flagellates

The flagellar apparatus of euglenoids consists of two functional basal bodies, three unequal microtubular roots subtending the reservoir, and a fourth band of microtubules nucleated from one of the flagellar roots and subtending the reservoir membrane. The flagellar apparatus of some euglenoids may contain additional basal bodies, striated roots ("rhizoplasts"), fibrous roots, striated connecting fibers between basal bodies, layered structures, or various electron-dense connective substances. With the possible exception of Petalomonas cantuscygni, nearly all euglenoids are biflagellate although the length of one flagellum may be highly reduced. The flagellar transition zone and number of basal bodies are highly variable among species. In recent years a cytoplasmic pocket that branches off from the reservoir has been discovered. The microtubules of the ventral flagellar root are continuous with the microtubules which line this pocket. Based on positional and structural similarities, this structure is believed to be homologous with the MTR/cytostome of bodonids. Coupled with other ultrastructural and biochemical data, the fine structure of the flagellar apparatus supports the belief that the euglenoid flagellates are descendant from bodonid ancestors.     

Visualization of intermediate filaments in living cells using fluorescently labeled desmin

Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.     

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.     

Cell types and evidences for traumatic cell death in a pressure-bearing tendon of Rana catesbeiana

Tendon fibrocartilages appear in areas subjected to compressive forces. The bullfrog plantaris longus tendon was shown to be subjected to compression and to develop a modified region which differs from fibrocartilage in many respects. Ultrastructural analyses of the compression region of the bullfrog tendon demonstrated the existence of typical fibroblasts in the fibrous areas and large cells with abundant cytoplasm filled with intermediate type filaments. This large cell type has organelles restricted to a small perinuclear area or dispersed in the network of intermediate type filaments. Other cells were also found and exhibited less abundant deposition of intermediate filaments, showing an organization intermediate between fibroblasts and typical cells from the compression region. These intermediate type cells are closely associated with collagen bundles while the large cells seemed to have no connection with the fibrous components, but are immersed in a glycosaminoglycan-rich extracellular matrix. Aspects of cell death in association with extracellular matrix disruption were observed in some instances and it is likely that these are associated with traumatic stimulation of the tendon, especially when it is submitted to the sudden and strong mechanical loading expected to occur during jumping. Since the damage occurred mainly in cells of the intermediate type, it is assumed that accumulating intermediate type filaments is a protective mechanism against compressive forces to which this tendon is subjected.     

Cell types and evidences for traumatic cell death in a pressure-bearing tendon of Rana catesbeiana

Tendon fibrocartilages appear in areas subjected to compressive forces. The bullfrog plantaris longus tendon was shown to be subjected to compression and to develop a modified region which differs from fibrocartilage in many respects. Ultrastructural analyses of the compression region of the bullfrog tendon demonstrated the existence of typical fibroblasts in the fibrous areas and large cells with abundant cytoplasm filled with intermediate type filaments. This large cell type has organelles restricted to a small perinuclear area or dispersed in the network of intermediate type filaments. Other cells were also found and exhibited less abundant deposition of intermediate filaments, showing an organization intermediate between fibroblasts and typical cells from the compression region. These intermediate type cells are closely associated with collagen bundles while the large cells seemed to have no connection with the fibrous components, but are immersed in a glycosaminoglycan-rich extracellular matrix. Aspects of cell death in association with extracellular matrix disruption were observed in some instances and it is likely that these are associated with traumatic stimulation of the tendon, especially when it is submitted to the sudden and strong mechanical loading expected to occur during jumping. Since the damage occurred mainly in cells of the intermediate type, it is assumed that accumulating intermediate type filaments is a protective mechanism against compressive forces to which this tendon is subjected.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

Ultrafine organization of Leptomonas peterhoffi flagellates cultured in broth and solid nutrient media

The morphology of in vitro grown lower trypanosomatids L. peterhoffi was studied by means of electron microscopy. The flagellates from both liquid and solid culture media are represented by uninucleate cells of two structural types. Type I flagellates are characterized by dense cytoplasm enriched with numerous ribosomes. Type II flagellates are most abundant in the cultures; they display a less dense cytoplasm and fewer ribosomes. The flagella of L. peterhoffi of both types form enlargements, which are most expressed at the outlet of the flagellar pocket. The nuclei of some cells contain twisted threads about 10 nm in diameter. L. peterhoffi from the liquid media usually possess long, narrow and curved flagellar pockets. On the solid medium, amoeboid and hemispherical colonies composed of both uninucleate and giant multinucleate cells are formed. In these cells the flagellar pockets are usually short and straight. Outside the flagellar pocket, the axoneme often becomes looped in the flagellar enlargements of the colonial uninucleate cells.     

Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Trypanosoma brucei BILBO1 (TbBILBO1) is an essential component of the flagellar pocket collar of trypanosomes. We recently reported the high resolution structure of the N-terminal domain of TbBILBO1. Here, we provide further structural dissections of its other three constituent domains: EF-hand, coiled coil, and leucine zipper. We found that the EF-hand changes its conformation upon calcium binding, the central coiled coil forms an antiparallel dimer, and the C-terminal leucine zipper appears to contain targeting information. Furthermore, interdimer interactions between adjacent leucine zippers allow TbBILBO1 to form extended filaments in vitro. These filaments were additionally found to condense into fibers through lateral interactions. Based on these experimental data, we propose a mechanism for TbBILBO1 assembly at the flagellar pocket collar.     

The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure

Common prokaryotic motility modes are swimming by means of rotating internal or external flagellar filaments or gliding by means of retracting pili. The archaeabacterial flagellar filament differs significantly from the eubacterial flagellum: (1) Its diameter is 10-14 nm, compared to 18-24 nm for eubacterial flagellar filaments. (2) It has 3.3 subunits/turn of a 1.9 nm pitch left-handed helix compared to 5.5 subunits/turn of a 2.6 nm pitch right-handed helix for plain eubacterial flagellar filaments. (3) The archaeabacterial filament is glycosylated, which is uncommon in eubacterial flagella and is believed to be one of the key elements for stabilizing proteins under extreme conditions. (4) The amino acid composition of archaeabacterial flagellin, although highly conserved within the group, seems unrelated to the highly conserved eubacterial flagellins. On the other hand, the archaeabacterial flagellar filament shares some fundamental properties with type IV pili: (1) The hydrophobic N termini are largely homologous with the oligomerization domain of pilin. (2) The flagellin monomers follow a different mode of transport and assembly. They are synthesized as pre-flagellin and have a cleavable signal peptide, like pre-pilin and unlike eubacterial flagellin. (3) The archaeabacterial flagellin, like pilin, is glycosylated. (4) The filament lacks a central channel, consistent with polymerization occurring at the cell-proximal end. (5) The diameter of type IV pili, 6-9 nm, is closer to that of the archaeabacterial filament, 10-14 nm. A large body of data on the biochemistry and molecular biology of archaeabacterial flagella has accumulated in recent years. However, their structure and symmetry is only beginning to unfold. Here, we review the structure of the archaeabacterial flagellar filament in reference to the structures of type IV pili and eubacterial flagellar filaments, with which it shares structural and functional similarities, correspondingly.     

Cryptobia udonellae sp. n. (Kinetoplastidea: Cryptobiida) - parasites of the excretory system of Udonella murmanica (Udonellida)

A new cryptobiid flagellates, Cryptobia udonellae sp. n., is described from the excretory channels of Udonella murmanica. The body of flagellates is spindle-shaped. The flagellar pocket is subapical. Two flagella emerge from the pocket. One flagellum turns anterior and is forward-directed; the other flagellum is directed posterior and close to the ventral cell surface. The ventral groove is well developed. The cytostome opens just anterior to the flagellar pocket. The cytostome leads to the short cytopharynx. In the excretory channel of worms the flagellates C. udonellae sp. n. are attached to microvilli of epithelium or lay free in the lumen. Both flagellates have been studied with TEM. The unusual parasite system which involves organisms of four different phylums of animals has been described for the first time.     

Fine structure of bovine lens epithelial cells in vitro in relation to modifications induced by a retinal extract (EDGF)

Cultured bovine lens epithelial cells are polygonal in shape. In confluent and multilayer cultures they exhibit elaborate arrays of 6 nm filaments, bundles of intermediate-sized filaments, and a fibrous meshwork of subcellular and intercellular material. Cells grown in the presence of a retinal extract (RE) have a higher growth rate, and are smaller and more regular in shape. In them the 6 nm filaments are mostly aligned in sheets, the intermediate-sized filaments form a fine network, and the cells are closely apposed to the plastic substratum. Some homogeneous material is formed intercellularly in older cultures. Cellular elongation, induced in the former cultures by the addition of RE, is accompanied by an alignment of cytoskeletal elements, including microtubules, parallel to the long axis. Other structural features are similar in all cell types. The response to RE is discussed in terms of shape modulations associated with the restricted expression of structural characteristics acquired in vitro.