Reorientation of the microtubule-organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by binding to lysable target cells

By immunofluorescence observations with cell couples of cloned murine cytotoxic T lymphocytes (CTL) and target cells, evidence is presented for a rapid reorientation of the microtubule-organizing center (MTOC) and the Golgi apparatus (GA) in the effector cell (but not in the target cell) toward the contact area with the target. The reorientation of the MTOC/GA and the cytotoxic activity of the CTL were inhibited reversibly by nocodazole, a microtubule-disrupting agent. In lectin-formed cell couples of CTL and neuraminidase-treated target cells, the MTOC in essentially all of the CTL was oriented toward the effector-target contact area of a lysable target cell, but was left randomly oriented with a nonlysable target cell. A similar random orientation of the effector-MTOC was also observed in cell couples of cloned natural killer cells and nonlysable targets. These findings indicate that the repositioning of the MTOC and the GA, which is shared by CTL and natural killer cells, is an essential and early event in the onset of the cytolytic mechanism. It is suggested that this reorientation serves the purpose of directing to the bound target cell secretory vesicles derived from the GA that contain cytotoxic substances.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. I. Hypertrophy and change of orientation of the Golgi apparatus.

The ultrastructure of cytolytic T lymphocytes adhered to the surface of target cells was investigated at different periods after start of interaction. Fifteen-minute incubation led to increase of number of Golgi apparatus cisternae and vacuoles. After 30 min incubation Golgi apparatus become oriented to the contact area. If several lymphocytes adhered to one target cell the Golgi apparatus of each of them was oriented toward the contact area. If one lymphocyte adhered simultaneously to two target cells its Golgi apparatus was oriented toward both target cells. Giant Golgi apparatus vacuoles were formed 30 to 60 min later and then moved to plasma membrane of lymphocyte and then the content of those vacuoles moved to the intercellular space between a cytolytic T lymphocyte and a target cell. The period required for the hypertrophy and change of orientation of Golgi apparatus is supposed to represent the “mobilization” step of a medium-sized and small killer lymphocyte.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. II. Morphogenesis of secretory granules and intracellular vacuoles.

“Secretory” granules, crystal-like structures, lysosema-like granules, ergastoplasmatic lamellae, electron-opaque ribosomes, and lipid vacuoles were detected in the cytoplasm of cytolytic T lymphocytes. After 30 to 60 min interaction with a target cell the fusion of “secretory” granules and crystal-like structures with lipids, mitochondrion degeneration, hypertrophy and reorientation of the Golgi apparatus toward the contact zone with a target cell were observed.     

A unique ball-shaped Golgi apparatus in the rat pituitary gonadotrope: its functional implications in relation to the arrangement of the microtubule network

In polarized exocrine cells, the Golgi apparatus is cup-shaped and its convex and concave surfaces are designated as cis and trans faces, functionally confronting the rough endoplasmic reticulum and the cell surface, respectively. To clarify the morphological characteristics of the Golgi apparatus in non-polarized endocrine cells, the investigators immunocytochemically examined its precise architecture in pituitary gonadotropes, especially in relation to the arrangement of the intracellular microtubule network. The Golgi apparatus in the gonadotropes was not cup-shaped but ball-shaped or spherical, and its outer and inner surfaces were the cis and trans faces, respectively. Centrioles were situated at the center of the Golgi apparatus, from which radiating microtubules isotropically extended to the cell periphery through the gaps in the spherical wall of the Golgi stack. The shape of the Golgi apparatus and the arrangement of microtubules demonstrated in the present study could explain the microtubule-dependent movements of tubulovesicular carriers and granules within the gonadotropes. Furthermore, the spherical shape of the Golgi apparatus possibly reflects the highly symmetrical arrangement of microtubule arrays, as well as the poor polarity in the cell surface of pituitary gonadotropes.     

Contact regions of cytotoxic T lymphocyte-target cell conjugates.

The binding of cytotoxic T lymphocytes to target cells was studied by ultrastructural and tracer techniques. It was found that binding was achieved through interaction of the microvilli of both cells and that only a relatively small proportion of the cell surface was involved. Short points of contact, averaging 1500 Å in length, were the main form of junction. Periodic substructures were observed in some of the contact points. The transfer of cytoplasmic content from effector to target cell and vice versa was investigated, but no fluorescein or ⁵¹Cr-labeled components were transferred during the interaction. Examination of cell organelle localization during the interaction revealed that microfilaments were the only cellular components which localized at the contact area; the well-developed Golgi apparatus of the cytotoxic lymphocytes was randomly distributed.     

Contractile Torque as a Steering Mechanism for Orientation of Adherent Cells

It is well established that adherent cells change their orientation in response to non-uniform substrate stretching. Most observations indicate that cells orient away from the direction of the maximal substrate strain, whereas in some cases cells also align with the direction of the maximal strain. Previous studies suggest that orientation and steering of the cell may be closely tied to cytoskeletal contractile stress but they could not explain the mechanisms that direct cell reorientation. This led us to develop a simple, mechanistic theoretical model that could predict a direction of cell orientation in response to mechanical nonuniformities of the substrate. The model leads to a simple physical mechanism -- namely the contractile torque -- that directs the cell toward a new orientation in response to anisotropic substrate stretching or substrate material anisotropy. A direction of the torque is determined by a dependence of the contractile stress on substrate strain. Model predictions are tested in the case of simple elongation of the substrate and found to be consistent with experimental data from the literature.     

Cytolytic T lymphocyte-associated antigen-4 and the TCR zeta/CD3 complex, but not CD28, interact with clathrin adaptor complexes AP-1 and AP-2.

The negative signaling receptor cytolytic T lymphocyte-associated Ag-4 (CTLA-4) resides primarily in intracellular compartments such as the Golgi apparatus of T cells. However, little is known regarding the molecular mechanisms that influence this accumulation. In this study, we demonstrate binding of the clathrin adaptor complex AP-1 with the GVYVKM motif of the cytoplasmic domain of CTLA-4. Binding occurred primarily in the Golgi compartment of T cells, unlike with AP-2 binding that occurs mostly with cell surface CTLA-4. Although evidence was not found to implicate AP-1 binding in the retention of CTLA-4 in the Golgi, AP-1 appears to play a role in shuttling of excess receptor from the Golgi to the lysosomal compartments for degradation. In support of this, increased CTLA-4 synthesis resulted in an increase in CTLA-4/AP-1 binding and a concomitant increase in the appearance of CTLA-4 in the lysosomal compartment. At the same time, the level of intracellular receptor was maintained at a constant level, suggesting that CTLA-4/AP-1 binding represents one mechanism to ensure steady state levels of intracellular CTLA-4 in T cells. Finally, we demonstrate that the TCR zeta/CD3 complex (but not CD28) also binds to AP-1 and AP-2 complexes, thus providing a possible link between these two receptors in the regulation of T cell function.     

Transmembrane chloride flux is required for target cell lysis but not for Golgi reorientation in cloned cytolytic effector cells. Golgi reorientation, N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase release, and delivery of the lethal hit are separable events in target cell lysis

Cell-mediated cytotoxicity can be inhibited by the replacement of chloride with ions that are incapable of passing through chloride channels or by the presence of stilbene disulfonate derivatives known to interfere with chloride flux. We show that the stilbene disulfonate (4,4-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) inhibits lysis of YAC-1 targets by the cloned cell line NKB61A2. Inhibition of lysis occurs on the level of the effector cell inasmuch as preincubation of effectors but not of targets interferes with subsequent lysis. Moreover, inhibition of chloride flux in the target does not interfere with target cell lysis by cytotoxic granules isolated from killer cells. Target cell binding takes place in the presence of DIDS or absence of external chloride, suggesting that events that follow target cell binding require chloride flux. We show that reorientation of the Golgi apparatus, which occurs subsequent to target cell binding in the effector cell, occurs under conditions that interfere with chloride flux. It is therefore suggested that events in the effector cell taking place subsequent to the Golgi apparatus reorientation reaction are inhibited and that delivery of the lethal hit is a stimulus-induced secretory event that requires transmembrane chloride flux. Delivery of the lethal hit is shown to be independent of the release of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) serine esterase, suggesting that cytolytic components and BLT serine esterase are likely packaged in different vesicles.     

Deterministic Mechanical Model of T-Killer Cell Polarization Reproduces the Wandering of Aim between Simultaneously Engaged Targets

T-killer cells of the immune system eliminate virus-infected and tumorous cells through direct cell–cell interactions. Reorientation of the killing apparatus inside the T cell to the T-cell interface with the target cell ensures specificity of the immune response. The killing apparatus can also oscillate next to the cell–cell interface. When two target cells are engaged by the T cell simultaneously, the killing apparatus can oscillate between the two interface areas. This oscillation is one of the most striking examples of cell movements that give the microscopist an unmechanistic impression of the cell's fidgety indecision. We have constructed a three-dimensional, numerical biomechanical model of the molecular-motor-driven microtubule cytoskeleton that positions the killing apparatus. The model demonstrates that the cortical pulling mechanism is indeed capable of orienting the killing apparatus into the functional position under a range of conditions. The model also predicts experimentally testable limitations of this commonly hypothesized mechanism of T-cell polarization. After the reorientation, the numerical solution exhibits complex, multidirectional, multiperiodic, and sustained oscillations in the absence of any external guidance or stochasticity. These computational results demonstrate that the strikingly animate wandering of aim in T-killer cells has a purely mechanical and deterministic explanation.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells.

Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Electron microscopic study of the interaction between cytolytic T-lymphocytes and target cells]

Thymocytes stimulated in vitro in a mixed culture was sorbed by centrifugation on the surface of target cells for electron microscope study of cytology of immune T-lymphocytes and early cytolysis periods. A well developed Golgi apparatus was revealed in the cytoplasm of lymphocytes; there was also accumulation of tubular structures 50 to 60 nm in diameter which communicated with the cysterns of the granular endoplasmic reticulum, with "descended" vesiculi and plasmatic membrane of lymphocyte. This membrane formed numerous contacts with the membrane of target cells thus producing closed clefts. Taking into consideration these data and also current views on the intertransformation of membranes and intracellular transport a hypothetic scheme of the mechanism involved in the cytolysis of the target cell by immune T-lymphocyte was put forward.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

The Golgi apparatus at the cell centre

In non-polarised mammalian cells, the Golgi apparatus is localised around the centrosome and actively maintained there. Microtubules and molecular motor activity are required for determining both the localisation and organisation of the Golgi apparatus. Other factors, however, also appear necessary for regulating both the static steady-state distribution of this organelle and its relationship with microtubule minus-end-anchoring activities of the centrosome. Several non-motor microtubule-binding proteins have now been found to be associated with the Golgi apparatus. Recent advances suggest that, in addition to important roles in cell motility, polarisation and differentiation, the interplay between Golgi apparatus and centrosome could participate in other physiological processes such as intracellular signalling, mitosis and apoptosis.     

Accumulation of a GPI-anchored protein at the cell surface requires sorting at multiple intracellular levels

Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositol-anchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady-state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high-pressure frozen and freeze-substituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo- and exocytosis in this organism, is 48-52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum --> Golgi complex --> cell surface) or three enrichment steps (endoplasmic reticulum --> Golgi complex --> endosomes --> cell surface), suggesting sorting between several membrane compartments.     

MITOSIS, CELL WALL AND FLAGELLA SYNTHESIS IN SYNCHRONIZED CULTURES OF THE PRASINOPHYTE, SCHERFFELIA DUBIA

Cell division occurs within the parental cell wall, yielding two progeny cells. Since Scherffelia dubia sheds all four flagella prior to cell division, the maturing progeny cells must regenerate new cell walls and flagella during and/or after cytokinesis. To better understand these processes, we have synchronized cell division in cultures of S. dubia and observed all stages of mitosis, cytokinesis, and progeny cell maturation, including flagella and cell wall formation, via DAPI staining of fixed cells, DIC microscopy of live cells embedded in agarose and standard TEM. Microscopical observations revealed the following sequence of events: 1) Golgi stacks divide during late interphase and immediately begin producing theca scales; 2) deflagellation and release of the parental cell wall from the plasma membrane occurs during early prophase; 3) synthesis of theca and flagella scales within the Golgi and/or scale reticulum continues throughout mitosis; 4) during cytokinesis, a coalescence of vesicles containing theca scales at the posterior end of the cell results in a cleavage furrow slightly diagonal to the cells' longitudinal axis (40 min); 5) post-mitotic nascent basal body formation and flagella elongation at the inherited basal bodies (and later at the mature nascent basal bodies) occurs concurrently with continued cell wall synthesis; 6) the cleavage furrow rotates into a transverse position (35 min); 7) reorientation of the nuclei results in a "head to tail" orientation of the maturing progeny cells; and 8) matured progeny cells emerge from the posterior end of the parental theca not before 8 hrs after the onset of mitosis.     

A model for the interplay of receptor recycling and receptor-mediated contact in T cells

Orientation of organelles inside T cells (TC) toward antigen-presenting cells (APC) ensures that the immune response is properly directed, but the orientation mechanisms remain largely unknown. Structural dynamics of TC are coupled to dynamics of T-cell receptor (TCR), which recognizes antigen on the APC surface. Engagement of the TCR triggers its internalization followed by delayed polarized recycling to the plasma membrane through the submembrane recycling compartment (RC), which organelle shares intracellular location with the TC effector apparatus. TCR engagement also triggers TC-APC interface expansion enabling further receptor engagement. To analyze the interplay of the cell-cell contact and receptor dynamics, we constructed a new numerical model. The new model displays the experimentally observed selective stabilization of the contact initiated next to the RC, and only transient formation of contact diametrically opposed to the RC. In the general case wherein the TC-APC contact is initiated in an arbitrary orientation to the RC, the modeling predicts that the contact dynamics and receptor recycling can interact, resulting effectively in migration of the contact to the TC surface domain adjacent to the submembrane RC. Using three-dimensional live-cell confocal microscopy, we obtain data consistent with this unexpected behavior. We conclude that a TC can stabilize its contact with an APC by aligning it with the polarized intracellular traffic of TCR. The results also suggest that the orientation of TC organelles, such as the RC and the effector apparatus, toward the APC can be achieved without any intracellular translocation of the organelles.     

High concentration of calcium ions in Golgi apparatus

The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6-NBD-ceramide,and then the single cells were examined by laser scanning confocal microscopy(LSCFM) for subcellular distributions of Ca^2 and the location of Golgi apparatus.In these cells,the intracellular Ca^2 were found to be highly concentrated in the Golgi apparatus.The changes of distribution of cytosolic high Ca^2 region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium,when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin,the fluorescence of the Golgi region decreased far less than that of the cytosol.Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.     

Reconstitution of amoeboid motility in vitro identifies a motor-independent mechanism for cell body retraction

Crawling movement in eukaryotic cells requires coordination of leading-edge protrusion with cell body retraction [1-3]. Protrusion is driven by actin polymerization along the leading edge [4]. The mechanism of retraction is less clear; myosin contractility may be involved in some cells [5] but is not essential in others [6-9]. In Ascaris sperm, protrusion and retraction are powered by the major sperm protein (MSP) motility system instead of the conventional actin apparatus [10, 11]. These cells lack motor proteins [12] and so are well suited to explore motor-independent mechanisms of retraction. We reconstituted protrusion and retraction simultaneously in MSP filament meshworks, called fibers, that assemble behind plasma membrane-derived vesicles. Retraction is triggered by depolymerization of complete filaments in the rear of the fiber [13]. The surviving filaments reorganize to maintain their packing density. By packing fewer filaments into a smaller volume, the depolymerizing network shrinks and thereby generates sufficient force to move an attached load. Our work provides direct evidence for motor-independent retraction in the reconstituted MSP motility system of nematode sperm. This mechanism could also apply to actin-based cells and may explain reports of cells that crawl even when their myosin activity is compromised.     

Patterns of cell polarity in the developing mouse limb

Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are exreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane.     

THE GOLGI APPARATUS IN CHICK CORNEAL EPITHELIUM: CHANGES IN INTRACELLULAR POSITION DURING DEVELOPMENT

The intracellular position of the Golgi apparatuses in the basal cell layer of the corneal epithelium in embryonic and hatched chicks has been studied in the light microscope by impregnating the Golgi apparatus with silver. During two distinct periods in development the Golgi apparatuses in the basal cells shift from an apical to basal position. Each of these periods correlates in time with the appearance of an acellular collagenous matrix beneath the epithelium. Examination of the basal epithelial cells in the electron microscope confirms the intracellular shifts in position of the Golgi apparatus. The results suggest that the Golgi apparatus shifts to the basal cell pole of the corneal epithelium in order to excrete connective tissue materials into the developing corneal stroma.