Massive calcium–activated endocytosis without involvement of classical endocytic proteins

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4–64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP2) synthesis: PIP2 substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5′-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP2 induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP2 when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP2 and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A2 inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4–64, into vesicles and loss of reversible FM 4–64 binding confirm that 40–75% of the cell surface is internalized. Ongoing MEND stops in 2–4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and β-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces

Electrical and thermal transport in carbon nanotubes: Improved nanotube-based materials and thermoelectric power in the Coulomb blockade regime

We have studied the electrical and thermal properties of single wall carbon nanotubes (SWNTs) both at the bulk and single molecule level. The first group of experiments aims to verify the zone-folding model for the thermal conductivity κ(T) of SWNTs. Because of the geometry of SWNTs, the phonon (as well as electronic) states are quantized. This quantization leads to an observed linear behavior of κ(T) below 40 K. By varying the average diameters of bulk samples, the temperature at which higher phonon subbands contribute shifts, further evidence for phonon quantization. The second set of experiments are aimed at studying the transport properties of nanotube based materials. These include highly aligned SWNT films; SWNT-epoxy composites and nanotube fibers. We observed improvement in κ of these materials compared to disordered films and pristine epoxy. We have also characterized the electrical and thermal transport in C60-filled SWNTs and have found that at high temperatures, their thermoelectric power (TEP) does not become negative compared to unfilled material. This behavior is proposed to be due to the C60 balls preventing oxygen from leaving the interior of the tubes. The last set of experiments focuses on the first measurements of the TEP of SWNT bundles and individual SWNTs. We present a novel device we have developed that allows us to measure the TEP directly of single molecules. Our first experiments on bundles reveal p-type behavior and a temperature dependence that is very similar to air-exposed bulk samples. We surmise that inter-rope contacts are not important in the total TEP of SWNT films. At the single molecule level, the TEP of a semiconducting nanotube has been measured in the Coulomb blockade regime. We observe TEP oscillations, the amplitude and period of which are consistent with the theory of the TEP of quantum dots. Also, as the tube is depleted of carriers, the oscillations grow larger and are centered around an offset value of the TEP. We attribute the offset to defects on the tube and the Schottky barriers at the contacts which contribute a negative thermopower. From the TEP offset, we are also able to estimate the size of the depleted regions in the nanotube

Electrical and thermal transport in carbon nanotubes: Improved nanotube-based materials and thermoelectric power in the Coulomb blockade regime

We have studied the electrical and thermal properties of single wall carbon nanotubes (SWNTs) both at the bulk and single molecule level. The first group of experiments aims to verify the zone-folding model for the thermal conductivity κ(T) of SWNTs. Because of the geometry of SWNTs, the phonon (as well as electronic) states are quantized. This quantization leads to an observed linear behavior of κ(T) below 40 K. By varying the average diameters of bulk samples, the temperature at which higher phonon subbands contribute shifts, further evidence for phonon quantization. The second set of experiments are aimed at studying the transport properties of nanotube based materials. These include highly aligned SWNT films; SWNT-epoxy composites and nanotube fibers. We observed improvement in κ of these materials compared to disordered films and pristine epoxy. We have also characterized the electrical and thermal transport in C60-filled SWNTs and have found that at high temperatures, their thermoelectric power (TEP) does not become negative compared to unfilled material. This behavior is proposed to be due to the C60 balls preventing oxygen from leaving the interior of the tubes. The last set of experiments focuses on the first measurements of the TEP of SWNT bundles and individual SWNTs. We present a novel device we have developed that allows us to measure the TEP directly of single molecules. Our first experiments on bundles reveal p-type behavior and a temperature dependence that is very similar to air-exposed bulk samples. We surmise that inter-rope contacts are not important in the total TEP of SWNT films. At the single molecule level, the TEP of a semiconducting nanotube has been measured in the Coulomb blockade regime. We observe TEP oscillations, the amplitude and period of which are consistent with the theory of the TEP of quantum dots. Also, as the tube is depleted of carriers, the oscillations grow larger and are centered around an offset value of the TEP. We attribute the offset to defects on the tube and the Schottky barriers at the contacts which contribute a negative thermopower. From the TEP offset, we are also able to estimate the size of the depleted regions in the nanotube

Bacterial analysis of air sample from the nursery ward of a tertiary hospital in Alabang, Muntinlupa City

Air sample was collected from the nursery ward of tertiary government hospital in Alabang, Muntinlupa City. Sampling was conducted using the Andersen Staplex Air Sampler in July 2013. The bacterial load was found to be 90 CFU/ m3 of air, calculated from flow rate of 2.35 m3/hour and sampling period of 20 minutes. Polymerase chain reaction using 16s rDNA universal primers produced 1.5 kb amplicons in 66 isolates. PCR amplicons were sequenced using the forward and reverse primers, and were identified using the Basic Local Alignment Search Tool of The National Center for Biotechnology Information. Results showed the presence of 14 genera belonging to 24 different species of bacteria. The 35 isolatesor 53% were identified to belong to the genus Staphylococcus. The eight species of Staphylococcusisolated in the study have been reported to be opportunistic and known to cause infections.Pseudomonas(5 isolates) and Acinetobacter(4 isolates) are also known to be associated with healthcare-acquired infections. The study shows that it is imperative for the hospital administration to reassess the infection control program and be very strict in implementing aseptic procedures like proper handling of equipment, hand washing, hygiene, etc

Massive Endocytosis Activated by Perturbing the Outer Plasmalemmal Monolayer

The roles played by lipids in endocytic processes are the subject of much ongoing debate. Using electrophysiological, optical, and ultrastructural methods, we describe massive endocytosis (MEND) of >50% of the plasmalemma in response to perturbing the outer plasmalemma monolayer of fibroblasts and cardiac myocytes by multiple means. Extracellular application of a bacterial sphingomyelinase causes MEND within seconds, and similar responses occur with the nonionic detergents, Triton X-100 and NP-40, proapoptotic drugs (e.g. edelfosine and tamoxifen), and an amphipathic phospholipase inhibitor, U73122. At the concentrations employed, the effective agents do not cause membrane permeability changes, and they are inactive from the cytoplasmic side. Ca transients that do not cause MEND decrease markedly the threshold concentrations of amphipaths that cause MEND, perhaps by generating a lipid catalyst of MEND. Noise analysis of NP-40 records suggests that the average vesicle size is initially small (<100nm). However, internalized vesicles evidently fuse rapidly, as horseradish peroxidase is found within seconds in large vacuoles and multi-lamellar bodies. These MEND responses do not require cytoplasmic ATP, Ca, or dynamins, and they can be repeated multiple times with reversal taking place over several minutes in the presence of ATP. For nonionic detergents, ongoing MEND stops within 2 to 4 seconds when detergent is removed. For dodecylsulfate and dodecylglucoside, MEND occurs only after detergent removal. These results suggest that endocytosis can be driven primarily by lipidic forces, possibly by lipid and protein partitioning into domains that pinch off to the cytoplasm as a result of line tension to their surround