Platelet cryopreservation using a trehalose and phosphate formulation

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.     

Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes

Red blood cells (RBCs) can be cryopreserved using glycerol as a cryoprotective agent, but one of the main disadvantages is the time-consuming deglycerolization step. Novel cryopreservation strategies for RBCs using nontoxic cryoprotective agents are urgently needed. The effect of DMPC, DOPC, and DPPC liposomes on survival of RBCs cryopreserved with trehalose and HES has been evaluated. DMPC caused hemolysis before freezing and affected RBC deformability parameters. DMPC treated RBCs displayed a strong increase in trehalose uptake compared to control cells, whereas DOPC treated liposomes only displayed a slight increase in trehalose uptake. High intracellular trehalose contents were observed after cryopreservation. The recovery of cells incubated with trehalose and liposomes, frozen in HES ranged between 92.6 and 97.4% immediately after freezing. Recovery values of RBCs frozen in HES, however, decreased to 66.5% after 96 h at 4°C compared to 77.5% for DOPC treated RBCs. The recovery of RBCs incubated and frozen in trehalose medium was 77.8%. After 96 hours post-thaw storage recovery of these cells was 81.6%. DOPC and DPPC treated RBCs displayed higher recovery rates (up to 89.7%) after cryopreservation in trehalose compared to control RBCs. Highest survival rates were obtained using a combination of trehalose and HES: 97.8% directly after thawing and 81.8% 96-h post-thaw. DOPC liposomes, trehalose and HES protect RBCs during cryopreservation in a synergistic manner. The advantage is that the protective compounds do not need to be removed before transfusion.     

High‐Resolution Membrane Capacitance Measurements for Studying Endocytosis and Exocytosis in Yeast

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell‐attached configuration of the patch‐clamp techniques we were able to resolve the elementary processes of endo‐ and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2–1 fF which translates to vesicle diameters of 90–200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo‐ and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.     

FRET sensor-based quantification of intracellular trehalose in mammalian cells

Trehalose acts as a stress protectant and an autophagy inducer in mammalian cells. The molecular mechanisms of action remain obscure because intracellular trehalose at micromolar level is difficult to quantitate. Here, we show a novel trehalose monitoring technology based on FRET. FLIPsuc90μ?1Venus sensor expressed in mammalian cells enables to quickly and non-destructively detect an infinitesimal amount of intracellular trehalose.     

Characterization of endocytic uptake of MK2‐inhibitor peptides

Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP‐1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP‐1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae‐mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin‐mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Preformed Ω-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells

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Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin‐11 (Syt11), a non‐Ca²⁺‐binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin‐mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin‐coated pits and bulk endocytosis‐like structures increase on the plasma membrane in Syt11‐knockdown neurons. Structural–functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.     

A novel assay to demonstrate an intersection of the exocytic and endocytic pathways at early endosomes

The mechanism of transport of membrane proteins from the trans-Golgi to the cell surface is still poorly understood. Previous studies suggested that basolateral membrane proteins, such as the transferrin receptor and the asialoglycoprotein receptor H1, take an indirect route to the plasma membrane via an intracellular, most likely endosomal intermediate. To define this compartment we developed a biochemical assay based on the very definition of endosomes. The assay is based on internalizing anti-H1 antibodies via the endocytic cycle of the receptor itself. Internalized antibody formed immune complexes with newly synthesized H1, which had been pulse-labeled with [(35)S]sulfate and chased out of the trans-Golgi for a period of time that was insufficient for H1 to reach the surface. Hence, antibody capture occurred intracellularly. Double-immunofluorescence labeling demonstrated that antibody-containing compartments also contained transferrin and thus corresponded to early and recycling endosomes. The results therefore demonstrate an intracellular intersection of the exocytic and endocytic pathways with implications for basolateral sorting.     

Soybean meal‐induced uptake block in Atlantic salmon Salmo salar distal enterocytes

Soybean meal‐induced enteritis was characterized by the disappearance of the supranuclear vacuoles (SNV) in the enterocytes of the distal intestine of Atlantic salmon Salmo salar. The loss of SNV was correlated with an endocytosis uptake block as shown with ferritin as a marker molecule.     

Measurement of trehalose loading of mammalian cells porated with a metal-actuated switchable pore

Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellular trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now become very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibroblasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore-forming alpha-hemolysin from Staphylococcus aureus. Manipulating the extracellular Zn(2+) concentration selectively opens and closes this pore ( approximately 2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography-mass spectroscopy (GC-MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC-MS method, we demonstrate that the switchable characteristics of H5 alpha-hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desiccation tolerance in mammalian cells.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian sperm

Previous data showed that complexin I, a SNARE regulatory protein, is localized in and/or around the acrosome and is necessary for the acrosome reaction in sperm. To understand how complexin I regulates the acrosome reaction, we used complexin-GST pulldown assays to identify interacting proteins. We showed that both complexins I and II bound mouse sperm dynamin 2. Dynamin 2 is a 100 kDa GTPase essential to many aspects of endocytosis but its potential role in exocytosis is unknown. Dynamin 2 is expressed in rat testis and widely expressed in other tissues; however, the function of dynamin 2 in germ cells is uncertain. Dynamin 2 protein was detected in mouse testis and was most abundant in or around the developing acrosome of spermatids. In addition, dynamin 2 was co-localized with complexin I in the acrosomal region of mammalian sperm. Its co-localization and interaction with complexin I suggest that dynamin 2 may play a role during acrosome formation and/or acrosomal exocytosis.     

Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides

Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides.     

Intersection of endocytic and exocytic systems in Toxoplasma gondii

Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome‐like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant‐like features suggest T. gondii endocytic trafficking involves transit through early and late endosome‐like compartments (ELCs) and potentially the trans‐Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin‐related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post‐invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host‐derived proteins, suggesting that DrpB is not required for parasite endocytosis.      

Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity‐dependent bulk endocytosis

Activity‐dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity‐dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine‐dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium‐dependent events such as activity‐dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity‐dependent dynamin I dephosphorylation was also arrested in EGTA‐treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE.

     

Strategies for the cryopreservation of microencapsulated cells

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300-400 microm) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60-80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80-85%), with the majority of the microcapsules remaining intact ( approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either post-thaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.