The formation of cortical actin arrays in human trabecular meshwork cells in response to cytoskeletal disruption

The cytoskeleton of human trabecular meshwork (HTM) cells is known to be altered in glaucoma and has been hypothesized to reduce outflow facility through contracting the HTM tissue. Latrunculin B (Lat-B) and Rho-associated protein kinase (ROCK) inhibitors disrupt the actin cytoskeleton and are in clinical trials as glaucoma therapeutics. We have previously reported a transient increase in HTM cell stiffness peaking at 90 min after Lat-B treatment with a return to pretreatment values after 270 min. We hypothesize that changes in actin morphology correlate with alterations in cell stiffness induced by Lat-B but this is not a general consequence of other cytoskeletal disrupting agents such as Rho kinase inhibitors. We treated HTM cells with 2 µM Lat-B or 100 µM Y-27632 and allowed the cells to recover for 30–270 min. While examining actin morphology in Lat-B treated cells, we observed striking cortical actin arrays (CAAs). The percentage of CAA positive cells (CPCs) was time dependent and exceeded 30% at 90 min and decreased after 270 min. Y-27632 treated cells exhibited few CAAs and no changes in cell stiffness. Together, these data suggest that the increase in cell stiffness after Lat-B treatment is correlated with CAAs.     

Brefeldin A induces callose formation in onion inner epidermal cells

Summary The antibiotic fungal toxin brefeldin A (BFA) causes synthesis of additional cell wall material in adult differentiated onion inner epidermal cells at concentrations of 5–30 g/ml. This tertiary wall contains callose and is layered on the secondary cellulosic wall in a time- and dose-dependent manner. Initially, callose is found in pit fields in the form of small vesicular patches. With time and dose, depositions grow in size and form large plugs invaginating into the cell, where the adjacent cytoplasm forms bulky accumulations and contains many organelles including endomembranes. Within the cytoplasm, BFA exerts the characteristic morphological effects on the secretory system including changes of the Golgi stacks, formation of large vesicles, and proliferation of dilated cisternae of the endoplasmic reticulum. Higher concentrations of BFA (60 g/ml) lead to disintegration of the Golgi apparatus; they have no effects on the cell wall, no callose synthesis occurs. We conclude from these observations that BFA has two independent targets in onion cells. BFA acts on the plasma membrane, hence operating as an elicitor of plant defense reactions and thus activates callose synthesis. BFA acts also on the membranes of the secretory system and influences budding and fusion of vesicles at the endoplasmic reticulum and at the dictyosomes. These two mechanisms occur in parallel, suggesting that the secretory system still can play its presumed role in callose synthesis. Only when dictyosomes are completely disintegrated, no more callose is formed.Abbreviations BFA Brefeldin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum - GS glucan synthetase Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Analysis of desmosomal intramembrane particle populations and cytoskeletal elements: Detergent extraction and freeze-fracture

Summary We have examined sections and freeze-fracture replicas of Triton X-100 detergent-extracted desmosomes from murine palatal epithelium. After extraction of lipids as well as soluble proteins, a cytoskeletal framework remained which consisted of intermediate filaments, microfilaments, and intact desmosomal skeletons. Traversing filaments, which link the intermediate filaments to large intramembrane particles of the P-face, appeared undisturbed within the desmosomal skeletons. Compared to unextracted controls, extracted specimens displayed P- and E-face desmosomal intramembrane particles which were more fully exposed. A broad range of sizes and shapes was apparent for the P-face associated particles. E-face particles, some of which were exposed for the first time, were more homogeneous and generally smaller. Statistical data gathered from a large sample of P- and E-face particle diameters disclosed significant differences among the populations of the two faces. Both fracture faces of extracted desmosomal domains displayed a residual surface upon which the exposed particles seemed to remain lodged. The newly revealed structural features are presented in an hypothetical molecular model which provides for both vertical and horizontal stabilization of desmosomal subcomponents. The model may ultimately be relatable to emerging biochemical characterization.     

Changes of the actin filament system in the green algaMicrasterias denticulata induced by different cytoskeleton inhibitors

Summary Two different techniques have been adapted forMicrasterias denticulata to depict the actin cytoskeleton of both untreated and inhibitor-treated developing cells: the quickstaining method, where the cells are fixed in a mixture of glutaraldehyde and formaldehyde followed by staining with phalloidin without embedding, and the methacrylate method, where the cells are also fixed by aldehydes and where the embedding medium is removed prior to incubation with an actin antibody. Both methods produce sufficient preservation and visualization of actin microfilaments (MFs) and confirm earlier observations on the presence of a cortical actin MF network in both the growing and the nongrowing semicell as well as of a basketlike MF arrangement around the migrating nucleus. The results show that a network of actin MFs is essential for the proper development of the young lobes ofM. denticulata. Early developmental stages expanding uniformly at the beginning of growth lack any netlike actin MF arrangement. The actin cytoskeleton in developing cells treated with the actin-targeting agents cytochalasin D and latrunculin B is markedly influenced. Cytochalasin D, which produces the most pronounced effects, causes a breakdown of the network of actin MFs, resulting in bright actin clusters as well as in short and abnormally thick actin fragments particularly in cortical cell regions. In latrunculin B-treated cells remnants of the former actin MF network are still visible, yet most of the actin cytoskeleton appears collapsed and is reduced to short filament pieces. The disturbance of the actin MF system visualized in the present study correlates with the severe morphological and ultrastructural changes occurring in desmid cells as a consequence of both drugs. The dinitroanilin herbicide oryzalin, known to deploymerize cytoplasmic microtubules, causes also an impairment of the actin cytoskeleton inM. denticulata though not sufficient to influence normal cell growth and differentiation.Abbreviations CB cytochalasin B - CD cytochalasin D - DMSO dimethyl sulfoxide - FA formaldehyde - GA glutaraldehyde - LAT-A latrunculin A - LAT-B latrunculin B - MFs microfilaments - MT microtubuleDedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Activities of the chaperonin containing TCP-1 (CCT): implications for cell cycle progression and cytoskeletal organisation.

The chaperonin containing TCP-1 (CCT) is required for the production of native actin and tubulin and numerous other proteins, several of which are involved in cell cycle progression. The mechanistic details of how CCT acts upon its folding substrates are intriguing: whilst actin and tubulin bind in a sequence-specific manner, it is possible that some proteins could use CCT as a more general binding interface. Therefore, how CCT accommodates the folding requirements of its substrates, some of which are produced in a cell cycle-specific manner, is of great interest. The reliance of folding substrates upon CCT for the adoption of their native structures results in CCT activity having far-reaching implications for a vast array of cellular processes. For example, the dependency of the major cytoskeletal proteins actin and tubulin upon CCT results in CCT activity being linked to any cellular process that depends on the integrity of the microfilament and microtubule-based cytoskeletal systems.     

Autonomous circular and radial motions of the nucleus inPleurenterium tumidum and their relation to cytoskeletal elements and the plasma membrane

Summary InPleurenterium tumidum the nucleus leaves its central position at the end of cell development and moves centrifugally towards the cortical cytoplasm of the isthmus area. It passes between the chloroplast lobes and starts to perform circular motions along the cell wall ring of the isthmus independently from other cell organelles and cytoplasmic streaming. This autonomous nuclear motion is a unique phenomenon in plant cells which is reported here for the first time. One turn of the nucleus which may occur either clockwise or counter-clockwise lasts an average of 60 minutes. The velocity of circular nuclear motion lies between 0.03 and 0.08 m per second and increases with increasing number of nuclear turns. The nucleus undergoes at least 12 but sometimes up to 70 turns and may change its direction of motion several times. When circular nuclear motion is finished the nucleus migrates centripetally towards the cell center where the next mitosis takes place.Ultrastructural studies demonstrate that a distinct arrangement of the plasma membrane forming a ring-shaped fold together with the adjacent isthmus system of microtubules (IS) serves as a hoop-like track for the nucleus during the stage of circular motion. The nucleus moves along this track by surrounding it in a deep furrow which develops parallel to its longitudinal axis at its cell wall facing side. The spatial arrangement of the plasma membrane fold and the nuclear furrow are only present during the stage of circular nuclear motion. No actin filaments seem to be involved in the nuclear circulations since the nucleus continues its circular motions after cytochalasin B (CB) treatment even at concentrations which arrest cytoplasmic streaming. Amiprophos-methyl (APM) leads to an inhibition of circular nuclear motion which resumes when the APM solution is removed. Microtubules appear to be primarily responsible also for both the radial nuclear motions as well as the anchoring of the nucleus in its central position. The meaning of circular and radial nuclear motions for thePleurenterium cell is not yet clear, a relation between the nuclear behavior and the inner cell architecture is discussed and compared to that of other desmids.     

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.     

Distinct fluorescent pattern of KAT1::GFP in the plasma membrane of Vicia faba guard cells

The organisation of membrane proteins into certain domains of the plasma membrane (PM) has been proposed to be important for signalling in yeast and animal cells. Here we describe the formation of a very distinct pattern of the K(+) channel KAT1 fused to the green fluorescent protein (KAT1::GFP) when transiently expressed in guard cells of Vicia faba. Using confocal laser scanning microscopy we observed a radially striped pattern of KAT1::GFP fluorescence in the PM in about 70% of all transfected guard cells. This characteristic pattern was found to be cell type and protein specific and independent of the stomatal aperture and the cytoskeleton. Staining of the cell wall of guard cells with Calcofluor White revealed a great similarity between the arrangement of cellulose microfibrils and the KAT1::GFP pattern. Furthermore, the radial pattern of KAT1::GFP immediately disappeared when turgor pressure was strongly decreased by changing from hypotonic to hypertonic conditions. The pattern reappeared within 15 min upon reestablishment of high turgor pressure in hypotonic solution. Evaluation of the staining pattern by a mathematical algorithm further confirmed this reversible abolishment of the radial pattern during hypertonic treatment. We therefore conclude that the radial organisation of KAT1::GFP depends on the close contact between the PM and cell wall in turgid guard cells. These results offer the first indication for a role of the cell wall in the localisation of ion channels. We propose a model in which KAT1 is located in the cellulose fibrils intermediate areas of the PM and discuss the physiological role of this phenomenon.     

Terminology and nomenclature of the cytoskeletal elements associated with the flagellar/ciliary apparatus in protists

Summary The terminology used to describe the cytoskeleton of protists is sometimes redundant and confusing. The independent origins of protistology from studies on algae, fungi, and protozoans led to these problems. Additionally, recent studies indicate that seemingly unrelated taxa form new, monophyletic groups. However, homologous structures that support monophyly are difficult to identify when the literature is published in journals restricted to specific fields or when the terminology is in duplicate or triplicate. The purpose of this paper is to establish clear definitions, identify synonyms, and indicate homologies where possible.     

The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function

The ability to harvest and maintain viable cells from mammalian tissues represented a critical advance in biomedical research, enabling individual cells to be cultured and studied in molecular detail. However, in these traditional cultures, cells are grown on rigid glass or polystyrene substrates, the mechanical properties of which often do not match those of the in vivo tissue from which the cells were originally derived. This mechanical mismatch likely contributes to abrupt changes in cellular phenotype. In fact, it has been proposed that mechanical changes in the cellular microenvironment may alone be responsible for driving specific cellular behaviors. Recent multidisciplinary efforts from basic scientists and engineers have begun to address this hypothesis more explicitly by probing the effects of ECM mechanics on cell and tissue function. Understanding the consequences of such mechanical changes is physiologically relevant in the context of a number of tissues in which altered mechanics may either correlate with or play an important role in the onset of pathology. Examples include changes in the compliance of blood vessels associated with atherosclerosis and intimal hyperplasia, as well as changes in the mechanical properties of developing tumors. Compelling evidence from 2-D in vitro model systems has shown that substrate mechanical properties induce changes in cell shape, migration, proliferation, and differentiation, but it remains to be seen whether or not these same effects translate to 3-D systems or in vivo. Furthermore, the molecular “mechanotransduction” mechanisms by which cells respond to changes in ECM mechanics remain unclear. Here, we provide some historical context for this emerging area of research, and discuss recent evidence that regulation of cytoskeletal tension by changes in ECM mechanics (either directly or indirectly) may provide a critical switch that controls cell function.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.     

Fibrillar and cytoskeletal substructure of tight junctions: Analysis of single-stranded tight junctions linking fibroblasts of the lamina fusca in hamster eyes

Summary The lamina fusca of the hamster eye contains layers of flattened, slightly overlapping fibroblasts. Thin sections of the overlapping margins reveal punctate, tight-junction-like membrane appositions associated with accumulation of cytoplasmic filaments, 5–7 nm in diameter. Intermediate filaments are present in the surrounding cytoplasm. A diffuse dense substance occurs in adjacent intercellular space. Freeze-fracture replicas show that the membrane appositions are mainly single-stranded tight junctions, each composed of two fibrils (micelles), and each continuous or nearly continuous around the fibroblastic perimeter. Fracturing characteristics of these junctions offer a unique opportunity to gain further insight into tight junctional morphology. When exposed, the fibrils adhere to the P-face, measure 9.2±0.3 nm in diameter, and are accompanied by a narrow band of membrane differing in texture from non-junctional membrane. Characteristically, the junctional fibrils themselves mark the deviation line along which fracture planes pass from one membrane of the junction to the other. This pattern exposes, over long distances, the P-face of one membrane on one side of this line and E-face of the adjacent membrane on the other. Analysis of any single junction over such distances reveals that the juxtaposition of the fibrils may gradually twist or undulate over a range of at least 180° within the two involved membranes. The fracture plane appears preferentially to pass between the two junctional fibrils; association of the cytoskeleton with junctional fibrils may govern this route of fracture. Cytoskeletal attachment appears to be to a single fibril and may alternate from one fibroblast to the next depending on which cytoplasmic leaflet is nearest a given fibril.Parts of this work have been presented at meetings of the Association for Research in Vision and Ophthalmology (Kelly and Hageman 1983) and the American Association of Anatomists (Hageman and Kelly 1984)     

Immunocytochemistry of contractile and cytoskeletal proteins in smooth muscle: Lowicryl,LR White,and polyvinylalcohol compared

Summary Three embedding media have been compared with respect to post-embedding immunolabeling of contractile and cytoskeletal antigens in aldehyde-fixed smooth muscle tissue: the methacrylate derivates lowicryl K4M (cured at –35 or 60°C) and LR White (cured at 0 or 60°C) and the water soluble resin, polyvinylalcohol (dried at 60°C). Measurements of intensity of labeling of ultrathin sections in the fluorescence microscope showed that five antigens (actin, myosin light chain, tropomyosin, filamin and vinculin) reacted more or less equally with their respective antibodies in all the embedding media, including those cured at 60°C. One antibody (anti-light meromyosin) reacted well only with polyvinylalcohol-embedded tissue. In contrast to the relative invariance of antibody reactivity between media clear differences in the preservation of ultrastructural integrity were observed. Embedding in polyvinylalcohol (dried at 60°C) and in Lowicryl (cured at –35°C) resulted in superior preservation as compared to Lowicryl or LR White cured at 60°C. Examples of uitrastructural immunocytochemistry with the antibodies against filamin and myosin light chain, using the immunogold staining procedure are presented: the sites of localization by these antibodies were the same with all the media tried. The relative merits of the different methods are discussed.Abbreviations EGTA Ethyleneglycol-bis(-amino ethyl ether)N,N,N,N-tetra acetic acid - PIPES 1,4-Piperazinediethanesulfonic acid - LR London Resin     

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.     

The Drosophila ACP65A cuticle gene: deletion scanning analysis of cis-regulatory sequences and regulation by DHR38

The regulatory sequences of the Drosophila ACP65A cuticle gene were analyzed in vivo in transgenic flies, using both fusion genes constructs and transposase-mediated deletions within a P element containing ACP65A regulatory sequences fused to the lacZ gene (deletion scanning). The sequences located between -594 and +161 are sufficient to confer both temporal and spatial expression specificities, indicating the presence of tissue-specific enhancers and response elements to hormone-induced factors. In addition, timing of expression and tissue-specificity appear to be controlled by distinct cis-regulatory elements, which suggests the existence of independent hormonal and tissue-specific signaling pathways. Gain and loss of function studies also implicate DHR38, the Drosophila homolog of the vertebrate NGFI-B-type nuclear receptors, as an important activator of the ACP65A gene.     

The fate of SPE B after internalization and its implication in SPEB-induced apoptosis

Summary After streptococcal pyrogenic exotoxin B (SPE B) induces apoptosis, its fate is unknown. Using confocal time-course microscopy at 37 °C, we detected green fluorescence 20 min after adding FITC-SPE B. Orange fluorescence, an indication of co-localization of SPE B with lysosomes which were labeled with a red fluorescent probe, was maximal at 40 min and absent by 60 min. SPE B was co-precipitated with clathrin, which is consistent with endocytotic involvement. Western blotting assay also indicated that uptake of SPE B was maximal at 40 min and disappeared after 60 min. However, in the presence of chloroquine, a lysosome inhibitor, the uptake of SPE B was not detectable. The disappearance of TCA-precipitated FITC-SPE B was parallel to the appearance of TCA soluble FITC-SPE B; in the presence of chloroquine, however, no SPE B degradation occurred. Chloroquine increased the level of SPE B-induced apoptosis by inhibiting the degradation of SPE B. These results suggest that the internalization and degradation of SPE B in cells may be a host defense system that removes toxic substances by sacrificing the exposed cells.     

The dorsal neural tube: A dynamic setting for cell fate decisions

The dorsal neural tube first generates neural crest cells that exit the neural primordium following an epithelial‐to‐mesenchymal conversion to become sympathetic ganglia, Schwann cells, dorsal root sensory ganglia, and melanocytes of the skin. Following the end of crest emigration, the dorsal midline of the neural tube becomes the roof plate, a signaling center for the organization of dorsal neuronal cell types. Recent lineage analysis performed before the onset of crest delamination revealed that the dorsal tube is a highly dynamic region sequentially traversed by fate‐restricted crest progenitors. Furthermore, prospective roof plate cells were shown to originate ventral to presumptive crest and to progressively relocate dorsalward to occupy their definitive midline position following crest delamination. These data raise important questions regarding the mechanisms of cell emigration in relation to fate acquisition, and suggest the possibility that spatial and/or temporal information in the dorsal neural tube determines initial segregation of neural crest cells into their derivatives. In addition, they emphasize the need to address what controls the end of neural crest production and consequent roof plate formation, a fundamental issue for understanding the separation between central and peripheral lineages during development of the nervous system. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 796–812, 2010.     

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time- and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.