Movement Of Granules Research Articles

Velocity fluctuation and self diffusion character in a dense granular sheared flow studied by discrete element method

The distribution of average velocities, fluctuation of velocities, regional definition, and granular self-diffusion characters in dense granular flows between sheared parallel plates are discussed. In order to study the above problems, we use computer-established discrete element model with an average solid fraction of 0.80. Theoretical results show that the average velocities decrease with increasing height, and are larger for the case of lower plate with greater velocity; the average velocities in y direction are close to 0 because there is no bulk motion in y direction. Flows of the lower plate with a greater velocity induce relatively greater fluctuation of velocities in the x and y directions, the fluctuation of velocities increases with the height and is larger in the area close to the upper plate. The flows consist of a “solid-like” area in the lower test region, but a “fluid-like” region in the upper, and an “oscillating” region in the middle of the channel. By tracking the movements of granules continually, variations of the mean-square self-diffusion relative displacements with square time are plotted, and the mean self-diffusion relative coefficients are determined. As the fluctuation and self-diffusion analysis directly reflect the macroscopic properties of granules and provide bases and references for researching the flow mechanisms of “dense granular sheared flows”.

2P182 神経と接着した膵島α細胞内顆粒動態の解析(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

2P182 神経と接着した膵島α細胞内顆粒動態の解析(12. 細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

A possible role of transglutaminase 2 in the nucleus of INS-1E and of cells of human pancreatic islets

Transglutaminase 2 (TG2) is a multifunctional protein with Ca2 +-dependent transamidating and G protein activity. Previously we reported that the role of TG2 in insulin secretion may involve cytoplasmic actin remodeling and a regulative action on other proteins during granule movement. The aim of this study was to gain a better insight into the role of TG2 transamidating activity in mitochondria and in the nucleus of INS-1E rat insulinoma cell line (INS-1E) during insulin secretion. To this end we labeled INS-1E with an artificial donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins of the nuclear/mitochondrial-enriched fraction were analyzed using two-dimensional electrophoresis and mass spectrometry. Many mitochondrial proteins involved in Ca2 + homeostasis (e.g. voltage-dependent anion-selective channel protein, prohibitin and different ATP synthase subunits) and many nuclear proteins involved in gene regulation (e.g. histone H3, barrier to autointegration factor and various heterogeneous nuclear ribonucleoprotein) were identified among a number of transamidating substrates of TG2 in INS-1E. The combined results provide evidence that a temporal link exists between glucose-stimulation, first phase insulin secretion and the action of TG on histone H3 both in INS-1E and human pancreatic islets.Biological significanceResearch into the role of transglutaminase 2 during insulin secretion in INS-1E rat insulinoma cellular model is depicting a complex role for this enzyme. Transglutaminase 2 acts in the different INS-1E compartments in the same way: catalyzing a post-translational modification event of its substrates. In this work we identify some mitochondrial and nuclear substrates of INS-1E during first phase insulin secretion. The finding that TG2 interacts with nuclear proteins that include BAF and histone H3 immediately after (2–5 min) glucose stimulus of INS-1E suggests that TG2 may be involved not only in insulin secretion, as suggested by our previous studies in cytoplasmic INS-1E fraction, but also in the regulation of glucose-induced gene transcription.

The Rac activator DOCK2 regulates natural killer cell–mediated cytotoxicity in mice through the lytic synapse formation

AbstractNatural killer (NK) cells play an important role in protective immunity against viral infection and tumor progression, but they also contribute to rejection of bone marrow grafts via contact-dependent cytotoxicity. Ligation of activating NK receptors with their ligands expressed on target cells induces receptor clustering and actin reorganization at the interface and triggers polarized movement of lytic granules to the contact site. Although activation of the small GTPase Rac has been implicated in NK cell–mediated cytotoxicity, its precise role and the upstream regulator remain elusive. Here, we show that DOCK2, an atypical guanine nucleotide exchange factor for Rac, plays a key role in NK cell–mediated cytotoxicity. We found that although DOCK2 deficiency in NK cells did not affect conjugate formation with target cells, DOCK2-deficienct NK cells failed to effectively kill leukemia cells in vitro and major histocompatibility complex class I–deficient bone marrow cells in vivo, regardless of the sorts of activating receptors. In DOCK2-deficient NK cells, NKG2D-mediated Rac activation was almost completely lost, resulting in a severe defect in the lytic synapse formation. Similar results were obtained when the Rac guanine nucleotide exchange factor activity of DOCK2 was selectively abrogated. These results indicate that DOCK2-Rac axis controls NK cell–mediated cytotoxicity through the lytic synapse formation.

LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity

AbstractSecretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network–derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.

Rapid activation receptor– or IL-2–induced lytic granule convergence in human natural killer cells requires Src, but not downstream signaling

AbstractNatural killer (NK) cells participate in host defense by surveying for and ultimately killing virally infected or malignant target cells. NK cell cytotoxicity is a tightly regulated process that proceeds stepwise from adhesion and activation to the secretion of preformed lytic granule contents onto a diseased or stressed cell. We previously characterized rapid dynein-dependent lytic granule convergence to the microtubule-organizing center (MTOC) as an early, prerequisite step in NK cell cytotoxicity. Although multiple activating receptors can trigger granule convergence, the specific signal or signals responsible remained unknown. Using live cell confocal microscopy, NK cell lytic granule movement after NK cell activation was captured and measured. Using inhibitors of common early signaling mediators, we show that Src kinases are required for lytic granule convergence, but downstream signals that promote actin rearrangement, MTOC polarization, and calcium mobilization are not. Exposure to interleukin 2 was also sufficient to induce lytic granule convergence, which required noncanonical Src-dependent signaling. Thus, NK cell lytic granule convergence, prompted by specific receptor-mediated and soluble cytokine signals, depends on a directly downstream early Src kinase–dependent signal and emphasizes the importance of this step in readying NK cells for cytotoxicity.

Activation of Lymphocyte Cytolytic Machinery: Where are We?

Target cell recognition by cytotoxic lymphocytes implies the simultaneous engagement and clustering of adhesion and activating receptors followed by the activation of an array of signal transduction pathways. The cytotoxic immune synapse represents the highly specialized dynamic interface formed between the cytolytic effector and its target that allows temporal and spatial integration of signals responsible for a defined sequence of processes culminating with the polarized secretion of lytic granules. Over the last decades, much attention has been given to the molecular signals coupling receptor ligation to the activation of cytolytic machinery. Moreover, in the last 10 years the discovery of genetic defects affecting cytotoxic responses greatly boosted our knowledge on the molecular effectors involved in the regulation of discrete phases of cytotoxic process at post-receptor levels. More recently, the use of super resolution and total internal reflection fluorescence imaging technologies added new insights on the dynamic reorganization of receptor and signaling molecules at lytic synapse as well as on the relationship between granule dynamics and cytoskeleton remodeling. To date we have a solid knowledge of the molecular mechanisms governing granule movement and secretion, being not yet fully unraveled the machinery that couples early receptor signaling to the late stage of synapse remodeling and granule dynamics. Here we highlight recent advances in our understanding of the molecular mechanisms acting in the activation of cytolytic machinery, also discussing similarities and differences between Natural killer cells and cytotoxic CD8+ T cells.

Acoustic detection of melanosome transport in Xenopus laevis melanophores

Organelle transport studies are often performed using melanophores from lower vertebrates due to the ease of inducing movements of pigment granules (melanosomes) and visualizing them by optical microscopy. Here, we present a novel methodology to monitor melanosome translocation (which is a light-sensitive process) in the dark using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. This acoustic sensing method was used to study dispersion and aggregation of melanosomes in Xenopus laevis melanophores. Reversible sensor responses, correlated to optical reflectance measurements, were obtained by alternating addition and removal of melatonin (leading to melanosome aggregation) and melanocyte-stimulating hormone (MSH) (leading to melanosome dispersion). By confocal microscopy, it was shown that a vertical redistribution of melanosomes occurred during the dispersion/aggregation processes. Furthermore, the transport process was studied in the presence of cytoskeleton-perturbing agents disrupting either actin filaments (latrunculin) or microtubules (nocodazole). Taken together, these experiments suggest that the acoustic responses mainly originate from melanosome transport along actin filaments (located close to the cell membrane), as expected based on the penetration depth of the QCM-D technique. The results clearly indicate the potential of QCM-D for studies of intracellular transport processes in melanophores.

NK cell lytic granules are highly motile at the immunological synapse and require F-actin for post-degranulation persistence.

The formation of a dynamic, actin-rich immunological synapse (IS) and the polarization of cytolytic granules toward target cells are essential to the cytotoxic function of NK cells. Following polarization, lytic granules navigate through the pervasive actin network at the IS to degranulate and secrete their toxic contents onto target cells. We examined lytic granule motility and persistence at the cell cortex of activated human NK cells, using high-resolution total internal reflection microscopy and highly quantitative analysis techniques. We illustrate that lytic granules are dynamic and observe substantial motility at the plane of the cell cortex prior to, but not after, degranulation. We also show that there is no significant change in granule motility in the presence of Latrunculin A (which induces actin depolymerization), when added after granule polarization, but that there is a significant decrease in lytic granule persistence subsequent to degranulation. Thus, we show that lytic granules are highly dynamic at the cytolytic human NK cell IS prior to degranulation and that the persistence of granules at the cortex following exocytosis requires the integrity of the synaptic actin network.

A Unique Ball-Shaped Golgi Apparatus in the Rat Pituitary Gonadotrope

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.

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase–activating protein Gem-interacting protein

Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase-activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin.

Transglutaminase 2 transamidation activity during first-phase insulin secretion: natural substrates in INS-1E

Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-dependent transamidating and G protein activity. Previously, we reported that tgm2 -/- mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2's transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca(2+)] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2's role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.

A snake of a different color: physiological color change in Arizona black rattlesnakes

Coloration may serve a variety of behavioral (e.g., crypsis, communication) and physiological (e.g., thermoregulation, protection) functions for terrestrial ectotherms. However, optimal coloration for a given function may vary over environments (spatial or temporal) or conflict with other functions. Physiological color change (rapid change due to movement of pigment granules within chromatophores) may be an adaptation to resolve conflicting selective pressures on coloration. The proximate factors related to physiological color change are well known in many animals, but few studies have investigated the ecological or evolutionary implications of this behavior. Here, we present alternative hypotheses for physiological color change and discuss biotic and abiotic factors that may have led to the maintenance and/or loss of this behavior among populations of Arizona black rattlesnakes (Crotalus cerberus). We discuss what is known about this behavior and propose to investigate the function and evolution of coloration and color change in C. cerberus.

A snake of a different color: physiological color change in Arizona black rattlesnakes

AbstractColoration may serve a variety of behavioral (e.g., crypsis, communication) and physiological (e.g., thermoregulation, protection) functions for terrestrial ectotherms. However, optimal coloration for a given function may vary over environments (spatial or temporal) or conflict with other functions. Physiological color change (rapid change due to movement of pigment granules within chromatophores) may be an adaptation to resolve conflicting selective pressures on coloration. The proximate factors related to physiological color change are well known in many animals, but few studies have investigated the ecological or evolutionary implications of this behavior. Here, we present alternative hypotheses for physiological color change and discuss biotic and abiotic factors that may have led to the maintenance and/or loss of this behavior among populations of Arizona black rattlesnakes (Crotalus cerberus). We discuss what is known about this behavior and propose to investigate the function and evolution of coloration and color change in C. cerberus.

Granzyme B‐tdTomato, a new probe to visualize cytolytic effector cell activation

Granzyme B‐tdTomato, a new probe to visualize cytolytic effector cell activation

Platelet Spreading Requires Exocytosis of a Subpopulation of α-Granules Expressing VAMP-7,

Abstract 3248There has been recent controversy as to whether platelet α-granules represent a single, homogenous granule population or are composed of different subpopulations that contain different cargos and serve discrete functions. To evaluate this question, we have studied granule movement in spreading platelets. During platelet spreading, the majority of dense and α-granules coalesce in the central granulomere. However, some granules are observed in the platelet periphery. We evaluated whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Granules in spread platelets were stained with antibodies directed at vesicle-associated membrane proteins (VAMPs) −3, −7, and −8. We and others have shown that VAMP-3 and −8 function in granule exocytosis. VAMP-7 contains a profilin-like N-terminal extension capable of interacting with Arp2/3. Samples were imaged using confocal microscopy. Quantitative evaluation of antibody staining in the granulomere vs. the periphery demonstrated that 93+/−7% of VAMP-8, 86+/−14% of von Willebrand factor (an α-granule cargo), and 93+/−7% of serotonin (a dense granule cargo) localized to the central granulomere. In contrast, 77+/−8% of VAMP-7 localized to the periphery. To determine whether a VAMP-7+ α-granule subpopulation actively moves from the granulomere to the platelet periphery during spreading, we loaded platelet α-granules with Qdot 565 nanocrystals and performed time-lapse video microscopy of single platelets. While the majority of endocytosed nanocrystals remained in the central granulomere during spreading, a subpopulation of labeled granules actively moved from the granulomere towards the growing edge at the platelet periphery. Colocalization studies demonstrated that 73% of peripheral Qdot 565 nanocrystals colocalized with VAMP-7. Furthermore, 82% of granules expressing VAMP-7 colocalized with P-selectin (specific for α-granules). These results indicate that a subpopulation of VAMP-7+ α-granules actively moves to the platelet periphery during spreading. To determine whether α-granules are required for spreading, we evaluated spreading in platelets from a patient with gray platelet syndrome (GPS, a congenital absence of α-granules). Platelet surface area increased 4-fold with spreading in normal controls. In contrast, the increase in surface area of GPS platelets following plating for 15 min was minimal – suggesting that α-granules are required for platelet spreading. We also evaluated spreading in murine Jinx platelets, which lack functional Munc13-4. These platelets demonstrate normal morphology and intact proximal signaling, but have a severe granule exocytosis defect. Total surface area of Munc13-4 deficient platelets spread for 15 min was 47+/−3% that of wild-type controls. Platelet spreading was also evaluated using a grated optical biosensor capable of detecting membrane contact with its surface in real time. This technique showed that spreading was decreased in Munc13-4 deficient platelets to 48+/−17% of controls. These results identify a new α-granule subtype that expresses VAMP-7 and is required for platelet spreading. This observation supports the premise that α-granules are heterogeneous and demonstrates that different VAMP isoforms localize to functionally discrete α-granule subpopulations. Disclosures:Michelson:GLSynthesis: Research Funding; Lilly/Daiichi Sankyo: Data Monitoring Committee for clinical trial, Research Funding; Takeda: Research Funding.

Ultrastructural and Real-time Microscopic Changes in P. falciparum-infected Red Blood Cells Following Treatment with Antimalarial Drugs

Ultrastructural changes to P. falciparum-infected red blood cells were examined in vitro after treatment with antimalarial drugs. Artesunate had the most rapid parasitocidal effect. All three drugs caused structural changes within the parasite, including dilatation of the parasitophorus vacuole membrane, depletion of ribosomes, mitochondrial swelling, and decreased formation of hemozoin crystals. The structure of surface knobs and Maurer’s clefts were similar to controls but reduced in number. Only depletion of free ribosomes correlated with antimalarial drug exposure. Drug treatment decreased movement of hemozoin granules within parasites on real-time microscopy, before recognizable morphological changes of parasite death.

Real-time detection of mast cell degranulation in anaphylactoid reaction

To establish a new, real time, dynamic and direct optical detection method for mast cell degranulation caused by anaphylactoid reaction. A CD63-GFP plasmid was constructed and introduced steadily into rat basophilic leukemia (RBL-2H3) cells. The movements of CD63-GFP, which was located on both the granule membranes and the plasma membranes of RBL cells stimulated by Compound 48/80, were studied by confocal laser scanning microscope (CLSM) and total internal reflection fluorescence microscope (TIRFM) both inside and on the surface of living RBL-2H3 cells. Before antigen stimulation, most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation, the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.05 micron x s(-1). Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The method is rapid, sensitive and reliable, which could be used as a new detection method for anaphylactoid reaction in vitro.

A model of cytoplasmically driven microtubule-based motion in the single-celled Caenorhabditis elegans embryo

We present a model of cytoplasmically driven microtubule-based pronuclear motion in the single-celled Caenorhabditis elegans embryo. In this model, a centrosome pair at the male pronucleus initiates stochastic microtubule (MT) growth. These MTs encounter motor proteins, distributed throughout the cytoplasm, that attach and exert a pulling force. The consequent MT-length-dependent pulling forces drag the pronucleus through the cytoplasm. On physical grounds, we assume that the motor proteins also exert equal and opposite forces on the surrounding viscous cytoplasm, here modeled as an incompressible Newtonian fluid constrained within an ellipsoidal eggshell. This naturally leads to streaming flows along the MTs. Our computational method is based on an immersed boundary formulation that allows for the simultaneous treatment of fluid flow and the dynamics of structures immersed within. Our simulations demonstrate that the balance of MT pulling forces and viscous nuclear drag is sufficient to move the pronucleus, while simultaneously generating minus-end directed flows along MTs that are similar to the observed movement of yolk granules toward the center of asters. Our simulations show pronuclear migration, and moreover, a robust pronuclear centration and rotation very similar to that observed in vivo. We find also that the confinement provided by the eggshell significantly affects the internal dynamics of the cytoplasm, increasing by an order of magnitude the forces necessary to translocate and center the pronucleus.