Population Of Microvesicles Research Articles

Neutrophil microvesicles and their role in disease.

Microvesicles are formed through shedding from the plasma membrane, a process shared by almost all human cells. Microvesicles are highly abundant and have been detected in blood, urine, cerebrospinal fluid, and saliva. They contain a library of cargo derived from their parental cell during formation, including proteases, micro-RNAs and lipids and delivery of this parental cell-derived cargo to other cells can alter target cell function and drive disease. Cell specific molecules on the surface of microvesicles, obtained during microvesicle formation, allows their parental cell to be identified and populations of microvesicles to be investigated for roles in the pathogenesis of various diseases. For instance, recent work by our group has identified a role for neutrophil microvesicles in atherosclerosis. Microvesicle profiles could in future be associated with certain diseases and act as a biomarker to allow for earlier diagnosis. This short review will discuss some of the processes central to all microvesicles before focusing on neutrophil microvesicles, their potential role in cardiovascular disease and the mechanisms that may underpin this.

Microvesicles from Human Immortalized Cell Lines of Endothelial Progenitor Cells and Mesenchymal Stem/Stromal Cells of Adipose Tissue Origin as Carriers of Bioactive Factors Facilitating Angiogenesis.

Endothelial progenitor cells (EPCs) and mesenchymal stem/stromal cells (MSCs) are associated with maintaining tissue homeostasis and tissue repair. Both types of cells contribute to tissue regeneration through the secretion of trophic factors (alone or in the form of microvesicles). This study investigated the isolation and biological properties of microvesicles (MVs) derived from human immortalized MSC line HATMSC1 of adipose tissue origin and EPC line. The human immortalized cell line derived from the adipose tissue of a patient with venous stasis was established in our laboratory using the hTERT and pSV402 plasmids. The human EPC line originating from cord blood (HEPC-CB.1) was established in our previous studies. Microvesicles were isolated through a sequence of centrifugations. Analysis of the protein content of both populations of microvesicles, using the Membrane-Based Antibody Array and Milliplex ELISA showed that isolated microvesicles transported growth factors and pro- and antiangiogenic factors. Analysis of the miRNA content of isolated microvesicles revealed the presence of proangiogenic miRNA (miR-126, miR-296, miR-378, and miR-210) and low expression of antiangiogenic miRNA (miR-221, miR-222, and miR-92a) using real-time RT-PCR with the TaqMan technique. The isolated microvesicles were assessed for their effect on the proliferation and proangiogenic properties of cells involved in tissue repair. It was shown that both HEPC-CB.1- and HATMSC1-derived microvesicles increased the proliferation of human endothelial cells of dermal origin and that this effect was dose-dependent. In contrast, microvesicles had a limited impact on the proliferation of fibroblasts and keratinocytes. Both types of microvesicles improved the proangiogenic properties of human dermal endothelial cells, and this effect was also dose-dependent, as shown in the Matrigel assay. These results confirm the hypothesis that microvesicles of HEPC-CB.1 and HATMSC1 origin carry proteins and miRNAs that support and facilitate angiogenic processes that are important for cutaneous tissue regeneration.

Ischemic Conditions Affect Rerouting of Tau Protein Levels: Evidences for Alteration in Tau Processing and Secretion in Hippocampal Neurons

The spreading of misfolded protein species contributes to the propagation of harmful mediators in proteinopathies, including Alzheimer's disease (AD). Cellular stress circumstances, such as abnormal protein accumulation or nutrient deprivation, elicit the secretion of soluble misprocessed proteins and insoluble aggregates via multiple mechanisms of unconventional secretion. One of them consists in the rerouting of autophagic vacuoles towards exocytosis, an unconventional type of autophagy mediated by caspase-3 activation under starvation. Ischemic injury is a starvation condition characterized by oxygen/nutrient deprivation, whose contribution in AD onset has definitely been endorsed. Thus, we investigated the effect of oxygen-glucose deprivation (OGD), an experimental condition mimicking cerebral ischemia, in search of alteration in Tau processing and secretion in hippocampal neurons primary cultures. Our results showed that OGD caused alterations in Tau phosphorylation and processing, paralleled by an induction of its secretion. Interestingly, together with caspase-3 activation, full-length (FL) and fragmented Tau forms were secreted by their own or through a heterogeneous population of microvesicles (MVs), including autophagosome marker LC3-positive vesicles. Accordingly, confocal microscopy revealed a partial colocalization of intracellular Tau and LC3. Summarizing, our findings indicate that OGD alters Tau intracellular levels and protein processing. Consequently, Tau clearance was stimulated through multiple mechanisms related to unconventional Tau secretion, including exophagy. However, the activation of this response represent a double edge sword, because it could contribute to the spreading of misfolded Tau, a neurodegeneration pathway in AD and other tauopathies.

Biological Effects of Different Extracellular Vesicles Population on Reversal of Marrow Cells Radiation Damage

We have shown that Human or mouse mesenchymal stem cell (MSC) derived vesicles separated by differential centrifugation (300g, 10kg &100kg) can reverse radiation whole bone marrow (WBM) damage in vitro on the hematopoietic cell line FDC-P1 and in vivo on recovery of stem cells and differentiated blood cells after whole body irradiation. The vesicle separation is a classic method for preparations of exosomes. However, this separation isolates the smaller vesicles, discarding larger vesicles. Biologic healing effects of larger vesicles with or without smaller vesicles have not been investigated. Here, we investigated the healing effects of 3 different preparations of vesicles: 10k pellet (microvesicles), 100-10k pellet (exosomes) & 100k pellet (no 10k spin).Three different fractions (10k pellet, 100-10k pellet &100k pellet) of murine/human MSC or murine WBM derived vesicles were isolated by differential centrifugation. FDC-P1 cells were exposed at 500cGy irradiation and cultured with or without the addition of 3 different preparations of murine WBM or human MSC-derived vesicles for 7 days. A significant increase in cell proliferation was observed after 3 different vesicle fractions treatments. However, exosomes were clearly inferior, microvesicles and the combined exosomes and microvesicles population were superior. The capacity of human MSC-derived vesicles of different fractions on reversal of murine bone marrow damage was also evaluated in vivo. Again, the combined fractions shown a significant increase donor chimerism in bone marrow at 6 month post-transplant with 5x's the level of engraftment compared to irradiation control. The 10k and 100-10k fractions showed intermediate healing. Exposure to vesicles in irradiated FDC-P1 cells downregulated phosphorylated H2AX, the DNA damage marker and decreased apoptosis marker, PARP cleavage part. We further investigated the effect of murine marrow MSC-derived vesicles on the gene expression in peripheral blood cells from radiation damaged mice. 28 genes were altered by irradiation, 22 showed partial or complete reversal of these alterations. The characterization of three different fractions of vesicles derived from murine bone marrow cells and human MSC were evaluated by miRNA realtime PCR array (total 750 miRNA). Several MSC-EV associated miRNA responsible for the reversal of radiation damage were evaluated. Our preliminary data shown that miR-221, miR654-3p and miR210-5p could partially reverse radiation damage in FDC-P1 cellsThere are different biological effects in different extracellular vesicles populations. For reversal of radiation toxicity the most effective vesicle population would include both smaller (exosomes) and larger vesicles (microvesicles).Acknowedgements: This work was support by the NIH grant 5UH2TR00080 DisclosuresNo relevant conflicts of interest to declare.

Embryonic Stem Cell-Derived Microvesicles: Could They be Used for Retinal Regeneration?

Mouse embryonic stem cells (mESCs) release into the medium in which they are cultured heterogeneous populations of microvesicles (mESMVs), important components of cell-cell communication, that transfer their contents not only to other stem cells but also to cells of other origins. The purpose of these studies was to demonstrate that ESMVs could be the signals that lead the retinal progenitor Müller cells to de-differentiate and re-entry the cell cycle, followed by differentiation along retinal lineages. Indeed, we found that ESMVs induce these processes and change Müller cells' microenvironment towards a more permissive state for tissue regeneration.

Abstract 558: Neutrophils-derived-microvesicle Could Play a Role in the Early Stage of Atherosclerosis

Background: Atherosclerosis is the most common underlying cause of cardiovascular disease. Many cells types are involved in the initiation and progression of atherosclerotic plaques but, until recently, neutrophils were not considered to be among them. Despite their rare detection in plaques, neutrophil depletion has been shown to reduce plaque formation and monocyte recruitment to the vessel wall. Hypothesis: Neutrophils may influence plaque initiation and progression through the formation of microvesicles. Aim: To determine the role of neutrophil-derived-microvesicles in the early stage of atherosclerosis. Methods and Results: Mouse peripheral blood neutrophils were isolated by negative magnetic separation. Following stimulation for 1h with fMLP (3x105M), supernatants were subjected to differential centrifugation (twice at 300g for 6 min followed by 20,000g for 20 mins) to isolate the microvesicle population. Microvesicles were quantified using flow cytometry calibrated with MegaMix and Spherocount beads. Apo E mice were fed a Western diet for 6 weeks and injected twice a week with PBS or neutrophil microvesicles (4.52x106 ± 3.05x105) isolated from control mice. After 6 weeks, blood was collected for neutrophil and microvesicle quantification. The aorta was dissected and stained by conventional procedures using Oil Red. Areas of plaque formation was determined using computer based imaging and semi-quantitative scoring. Injection with microvesicles increased circulating neutrophil number (PBS treated = 67.3 ± 3.9 neutrophils per μl; microvesicle treated = 88.25 ± 4.0 neutrophils per μl, P = 0.031; n = 3) and neutrophil reactivity with respect to microvesicle formation (PBS treated = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle treated = 8.3±0.7 microvesicles per neutrophil, P = 0.0249; n = 3). After 6 weeks, the plaque formation was more advanced in the MVs group (% lesion area in the arch in mice injected with PBS = 0.4563 ± 0.09452, with MVs = 1.643 ± 0.3891, P = 0.0118, n=7). Conclusion: Neutrophil microvesicles induce increased plaque formation and increase circulating neutrophil levels in a mouse model of atherosclerosis. This suggests neutrophils may be involved in atherogenesis through the release of microvesicles.

Quantitative proteomics analysis of platelet-derived microparticles reveals distinct protein signatures when stimulated by different physiological agonists.

Platelet-derived MPs (PMPs) are a population of vesicles generated upon platelet activation by various stimuli known to be involved in several physiological and pathological processes. This manuscript investigates the protein profile of PMPs obtained by performing four different activation protocols using mass spectrometry-based quantitative proteomics. By following a known physiological agonist strength scale our findings suggest a biological link between agonist strength and proteins associated to platelet mediated processes such as activation and degranulation. These data may provide new insights for understanding PMP biological role and formation.

Sphingomyelin derivatives increase the frequency of microvesicle and granule fusion in chromaffin cells

Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles.

Epididymosomes: a heterogeneous population of microvesicles with multiple functions in sperm maturation and storage.

Extracellular microvesicles present in the epididymal fluid have been named epididymosomes. Many epididymosome-associated proteins are transferred to spermatozoa during their maturation in the excurrent duct. Epididymosomes are heterogeneous, with their size varying between 50 and 250 nm. Two distinct population of epididymosomes characterized by different protein compositions and diameters have been isolated from the bovine epididymal fluid using different centrifugation protocols. One subpopulation of epididymosomes was characterized by CD9 and other tetraspanin partners. Transfer of proteins from these epididymosomes to maturing spermatozoa in co-incubation experiments was inhibited by antibodies against tetraspanin proteins. This suggests that this subpopulation of epididymosomes is involved in the acquisition of proteins involved in maturation by spermatozoa in the epididymis. The other population of epididymosomes was characterized by ELSPBP1 (epididymal sperm binding protein 1), known for its affinity for the phospholipid choline group. Flow cytometric analyses showed that ELSPBP1-positive epididymosomes only interacted with dying or dead epididymal spermatozoa in a Zn2 +-dependent manner. BLVRA (biliverdin reductase) was identified as a partner of ELSPBP1. This enzyme reduces biliverdin to bilirubin: two molecules with powerful anti-oxidant properties. We hypothesize that BLVRA is involved in an ROS-scavenging mechanism protecting live epididymal spermatozoa against detrimental molecules (ROS) released by dying cells. Therefore, it appears that there are at least two epididymosome population with distinct functions: targeting specific proteins to transiting spermatozoa by tetraspanin-mediated membrane fusion, and protection of epididymal spermatozoa against ROS released from dying cells. Further work is needed to understand functions of epididymosomes in epididymal physiology and sperm maturation and storage.

Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell–cell communication wherein vesicle uptake can induce a change in the recipient cellʼs state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations.

Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer.

Abstract 4880: Regulation of extracellular microvesicles secretion by Rab27b in glioblastoma multiforme

Abstract Background: Glioblastoma multiforme (GBM) is the most malignant form of astrocytomas and they release a heterogeneous population of microvesicles (MVEs) to the extracellular space. These MVEs are involved in intercellular communication by carrying oncogenes, and miRNAs that contribute with the tumor aggressiveness. However, the mechanisms of MVEs biogenesis and secretion in GBM are poorly understood. Material and Methods: Using five glioblastoma cell lines (LN-18, LN-229, U87MG, A-172 and U118MG) the vesicular biogenesis was characterize analyzing the expression of markers involved in steps of this process by western blotting. MVEs were also quantified using Nanoparticle Tracking Analysis (NTA - NanoSight®). Immunofluorescence was performed in these cell lines to characterize the cellular distribution of Rab27b, which is responsible for driving the multivesicular bodies to the plasma membrane. Immunohistochemistry (IHC) measured Rab27b expression in a tissue microarray with 60 GBMs samples and 13 non-neoplastic human brain tissues used as controls. IHC intensity was scored from 1 to 3 (1 weak; 2, moderate; 3, strong) and positive cells, from 0 to 3 (0, 0%; 1, 1-10%+; 2, 11-50%+; 3, ≥ 51%+). The final IHC score was represented by intensity + positivity. Student's t-test was performed to compare Rab27b expression in GBM and non neoplastic tissues. Kaplan-Meier and log-rank tests were used to correlate overall survival with Rab27b expression. Results: The cell lines showed a heterogeneous expression of markers involved in vesicle biogenesis. LN-18 cells presented higher levels of Cavelin-1, Flotillin -1, Alix and VPS4, p<0.01, but lower levels of Rab27b, p<0.0001. On the other hand, the U87MG cells standed out by higher levels of Rab27b, compared to the other cell lines studied, p<0.0001. Interesting, our results pointed that MVEs secretion is directed correlated to the expression pattern of Rab27b in GBM cells. Indeed, LN-18 cells showed lower levels of MVEs secretion (p<0.001) while U87MG cells presented higher MVEs secretion, p<0.001. In addition, Rab27b and CD63 (MVEs marker) were retained in the perinuclear region of LN -18 cells while in U87MG cells both proteins were distributed throughout the cytoplasm. Finally, Rab27b is overexpressed in GBM samples when compared to non-neoplastic tissue and patients presenting high expression of Rab27b showed lower survival outcomes. Conclusions: Our findings suggest that Rab27b may be crucial to the secretion of MVEs process in GBM. Furthermore, the overexpression of Rab27b in glioblastoma patients coincides with a poor prognostic phenotype. Supported by São Paulo State Foundation (FAPESP). Citation Format: Bruna R. Rodrigues, Fernanda S. Giudice, Marcos S. Dias, Antuani R. Baptistella, Rafael R. Malagoli, Paulo I. Sanematsu, Sergio Suzuki, Mariana Reis, Vilma R. Martins. Regulation of extracellular microvesicles secretion by Rab27b in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4880. doi:10.1158/1538-7445.AM2014-4880

Size, Morphology, and miRNA Abundance of Cell-Secreted Microvesicles

Cell-secreted microvesicles consist of two populations, exosomes and shedding vesicles, that are released from all cell types in response to specific stimuli, but by entirely different mechanisms. Exosomes and shedding vesicles both contain miRNAs, although at different compositions. A feature that is thought to distinguish exosomes from shedding vesicles is their rich repertoire of miRNA. However, individual contributions from these two populations of microvesicles in transmitting differentiation-regulating signals to target cells have not been established. Asymmetric flow field fractionation-multi angle light scattering is used to fractionate the two microvesicle populations into exosomes and larger microvesicles, characterize their size distributions, and estimate absolute particle numbers in each population. The different fractions are further characterized based on their morphologies using cryo-transmission electron microscopy. In addition, qRT-PCR is used to quantify the copy numbers for selected miRNAs in the two fractions. Microvesicles secreted from different cancer cell lines in response to serum deprivation contain different populations of exosomes and larger microvesicles with the exosomes typically an order of magnitude greater in particle numbers than the larger microvesicles. The particle numbers in both populations depend on the response time. Using miR-21 as a reporter for miRNAs, we find miR-21 in both microvesicle populations, but at significantly different concentrations. Moreover, the number of secreted microvesicles is significantly greater than the total copy numbers of miRNA for all cell lines. We conclude that significant numbers of exosomes and larger microvesicles are released from cancer cell lines in response to serum deprivation, but only small numbers of these secreted microvesicles contain miRNA, suggesting the mechanism for microvesicle-mediated miRNA transfer to target cells is stochastic in nature.

Microparticles: a component of various diseases

Microparticles (MPs) are phenotypically and functionally heterogeneous population of microvesicles. Although MP formation represents a physiological phenomenon. A multitude of pathologies, including inflammatory and autoimmune diseases, atherosclerosis, and malignancies, are associated with a considerable increase in circulating MPs. Elevated levels of platelet‑, endothelial cell‑, and monocyte‑derived MPs have been documented in a number of clinical conditions in which vascular dysfunction and inflammation are important pathophysiological mechanisms (e.g., coronary artery disease or thrombotic microangiopathies). Knowledge of the functional properties of MPs will contribute to a better understanding of the pathological mechanisms of communication between cells and of the causes of various diseases.

Optical and non-optical methods for detection and characterization of microparticles and exosomes.

Microparticles and exosomes are cell-derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport. Despite increasing scientific and clinical interest, no standard procedures are available for the isolation, detection and characterization of microparticles and exosomes, because their size is below the reach of conventional detection methods. Our objective is to give an overview of currently available and potentially applicable methods for optical and non-optical determination of the size, concentration, morphology, biochemical composition and cellular origin of microparticles and exosomes. The working principle of all methods is briefly discussed, as well as their applications and limitations based on the underlying physical parameters of the technique. For most methods, the expected size distribution for a given microvesicle population is determined. The explanations of the physical background and the outcomes of our calculations provide insights into the capabilities of each method and make a comparison possible between the discussed methods. In conclusion, several (combinations of) methods can detect clinically relevant properties of microparticles and exosomes. These methods should be further explored and validated by comparing measurement results so that accurate, reliable and fast solutions come within reach.

Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa

The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 microm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 microm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 microm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves.

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.

Intact platelet membranes, not platelet-released microvesicles, support the procoagulant activity of adherent platelets.

The possibility that platelets release microvesicles on adherence to either von Willebrand factor (vWf) or collagen was examined by flow cytometry analysis of the supernatant above layers of adherent platelets. No microvesicle release was detected as a result of adherence to vWf or to collagen, a known platelet agonist. Approximately 8% of the total platelet mass was released as microvesicles after thrombin stimulation of the vWf- or collagen-adherent platelets. A larger portion of the vWf-adherent platelet membranes (approximately 21%) was released as microvesicles subsequent to platelet stimulation with the nonphysiological agonist calcium ionophore A23187. Calpeptin, a calpain inhibitor, had no effect on microvesicle release, suggesting that calpain proteolysis of platelet cytoskeletal proteins was not responsible for microvesicle shedding under the conditions studied. Examination of the vWf-adherent platelets by scanning electron microscopy showed that virtually no microvesicles bound to exposed vWf multimers. No microvesicle binding to the adherent platelets was observed, indicating that the majority of the microvesicles were shed from the platelet and vWf surface on platelet activation. The ability of the microvesicle population to support procoagulant activity was measured with a prothrombinase activity assay and was compared with the activity supported by the adherent platelet membranes. More than 85% of the total prothrombinase activity remained associated with the adherent platelet membranes, both for unstimulated platelets and platelets stimulated with physiological agonists. Furthermore, the residual activity found in the buffer fraction containing detached platelets and any released microvesicles could be attributed to the detached platelets. No activity could be attributed to the microvesicles, as thrombin stimulation of either vWf-or collagen-adherent platelets did not promote increased procoagulant activity relative to the unstimulated adherent platelets, even though microvesicle release was detected as a result of agonist addition. Neither full platelet activation nor microvesicle shedding played an essential role in generating procoagulant activity in the adherent platelet system.

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

An efficient preparative isopycnic flotation method for the isolation of chitosomes

Chitin synthetase, a key enzyme in fungal cell wall biosynthesis, is located in chitosomes (microvesicles). To produce large quantities of chitosomes for immunochemical and biochemical characterization, we developed a two-step purification procedure in which isopycnic sucrose density gradients were centrifuged at ultra-high gravitational forces (fixed-angle rotor at 361,000×g Rav). Chitosomes from yeast cells ofMucor rouxii were separated from the soluble proteins and from the larger membranes by isopycnic centrifugation of the cell-free extract. The resulting crude chitosome sample was adjusted to a higher sucrose concentration, and a sucrose gradient was layered over the sample. Upon recentrifugation, the chitosomes moved up into the gradient and equilibrated at their buoyant density (1.15–1.16). This accelerated flotation separated contaminating particles of higher buoyant density (larger vesicles, ribosomes, and other miniorganelles) and yielded a large population of microvesicles with a mean diameter of 48.9±13.8 nm. This preparation contained vesicles essentially free of other particulate contaminants; more than 99% of the vesicles were smaller than 100 nm. When required, an additional velocity centrifugation step was added to remove the larger vesicles from the chitosome samples. This streamlined method for chitosome isolation was much simpler and faster than earlier isolation procedures, gave a high yield of functional chitosomes, and made the large scale isolation of these organelles possible.