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A Kusumi

Publications and source records attributed to A Kusumi.

At least 19 recordsLinked to original sources
PubMed1995

Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether.

Our previous results indicated that the plasma membrane of cultured normal rat kidney fibroblastic cell is compartmentalized for diffusion of receptor molecules, and that long-range diffusion is the result of successive intercompartmental jumps (Sako, Y. and Kusumi, A. 1994. J. Cell Biol. 125:1251-1264). In the present study, we characterized the properties of intercompartmental boundaries by tagging transferrin receptor (TR) with either 210-nm-phi latex or 40-nm-phi colloidal gold particles, and by dragging the particle-TR complexes laterally along the plasma membrane using laser tweezers. Approximately 90% of the TR-particle complexes showed confined-type diffusion with a microscopic diffusion coefficient (Dmicro) of approximately 10(-9) cm2/s and could be dragged past the intercompartmental boundaries in their path by laser tweezers at a trapping force of 0.25 pN for gold-tagged TR and 0.8 pN for latex-tagged TR. At lower dragging forces between 0.05 and 0.1 pN, particle-TR complexes tended to escape from the laser trap at the boundaries, and such escape occurred in both the forward and backward directions of dragging. The average distance dragged was half of the confined distance of TR, which further indicates that particle-TR complexes escape at the compartment boundaries. Since variation in the particle size (40 and 210 nm, the particles are on the extracellular surface of the plasma membrane) hardly affects the diffusion rate and behavior of the particle-TR complexes at the compartment boundaries, and since treatment with cytochalasin D or vinblastin affects the movements of TR (Sako and Kusumi as cited above), argument has been advanced that the boundaries are present in the cytoplasmic domain. Rebound of the particle-TR complexes when they escape from the laser tweezers at the compartment boundaries suggests that the boundaries are elastic structures. These results are consistent with the proposal that the compartment boundaries consist of membrane skeleton or a membrane-associated part of the cytoskeleton (membrane skeleton fence model). Approximately 10% of TR exhibited slower diffusion (Dmicro approximately 10(-10)-10(-11) cm2/s) and binding to elastic structures.

Animals↗
PubMed1994

Hydrophobic barriers of lipid bilayer membranes formed by reduction of water penetration by alkyl chain unsaturation and cholesterol.

The hydrophobicity profiles across phosphatidylcholine (PC)-cholesterol bilayer membranes were estimated in both frozen liposome suspensions and fluid-phase membranes as a function of alkyl chain length, unsaturation, and cholesterol mole fraction. A series of stearic acid spin labels, with the probe attached to various positions along the alkyl chain, cholesterol-type spin labels (cholestane and androstane spin labels), and Tempo-PC were used to examine depth-dependent changes in local hydrophobicity, which is determined by the extent of water penetration into the membrane. Local hydrophobicity was monitored primarily by observing the z component of the hyperfine interaction tensor (Az) of the nitroxide spin probe in a frozen suspension of the membrane at -150 degrees C and was further confirmed in the fluid phase by observing the rate of collision of Fe(CN)6(3-) with the spin probe in the membrane using saturation recovery ESR. Saturated-PC membranes show low hydrophobicity (high polarity) across the membrane, comparable to 2-propanol and 1-octanol, even at the membrane center where hydrophobicity is highest. Longer alkyl chains only make the central hydrophobic regions wider without increasing the level of hydrophobicity. Introduction of a double bond at C9-C10 decreases the level of water penetration at all locations in the membrane, and this effect is considerably greater than the cis configuration than with the trans configuration. Incorporation of cholesterol (30 mol %) dramatically changes the profiles; it decreases hydrophobicity (increases water penetration) from the polar headgroup region to a depth of approximately C7 and C9 for saturated- and unsaturated-PC membranes, respectively, which is about where the bulky rigid steroid ring structure of cholesterol reaches in the membrane. Membrane hydrophobicity sharply increases at these positions from the level of methanol to the level of pure hexane, and hydrophobicity is constant in the inner region of the membrane. Thus, formation of effective hydrophobic barriers to permeation of small polar molecules requires alkyl chain unsaturation and/or cholesterol. The thickness of this rectangular hydrophobic barrier is less than 50% of the thickness of the hydrocarbon regions. Results obtained in dioleoyl-PC-cholesterol membranes in the fluid phase are similar to those obtained in frozen membranes. These results correlate well with permeability data for water and amino acids in the literature.

Alkylation↗
PubMed1994

Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique.

Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1994

Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis.

Movements of transferrin and alpha 2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced contrast optical microscopy with 1.8 nm spatial precision and 33 ms temporal resolution by labeling the receptors with the ligand-coated nanometer-sized colloidal gold particles. For both receptor species, most of the movement trajectories are of the confined diffusion type, within domains of approximately 0.25 microns2 (500-700 nm in diagonal length). Movement within the domains is random with a diffusion coefficient approximately 10(-9) cm2/s, which is consistent with that expected for free Brownian diffusion of proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains at an average frequency of 0.034 s-1 (the residence time within a domain approximately 29 s), indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that long-range diffusion is the result of successive intercompartmental jumps. The macroscopic diffusion coefficients for these two receptor molecules calculated on the basis of the compartment size and the intercompartmental jump rate are approximately 2.4 x 10(-11) cm2/s, which is consistent with those determined by averaging the long-term movements of many particles. Partial destruction of the cytoskeleton decreased the confined diffusion mode, increased the simple diffusion mode, and induced the directed diffusion (transport) mode. These results suggest that the boundaries between compartments are made of dynamically fluctuating membrane skeletons (membrane-skeleton fence model).

Animals↗
PubMed1994

[Interleukin 3 (IL-3)].

Interleukin 3 (IL-3) was initially described in the supernates of cultures of viral-infected murine spleen cells, as a cytokine produced by T lymphocytes can promote differentiation of immature T lymphocytes. Later, it was found that IL-3 exhibited a striking effect on hematopoiesis. The recombinant molecule of murine and human IL-3 can promote the sustained proliferation of clones of mast cells and basophils. It also acts as a colony stimulating factor (CSF) for bone marrow cells. Although other CSFs generally stimulate specific lineages of myeloid or erythroid cells, IL-3 stimulates bone marrow to induce proliferation of a variety of clonal cell populations, including colonies of granulocytes, macrophages, megakaryocytes, eosinophils, basophils, mast cells, normoblasts and erythroblasts. Thus, IL-3 is responsible for promoting proliferation of earlier lineage pluripotent stem cells, of hematopoietic cells and lymphoid cells. Recently, it is also suggested, as to its effects on lymphocytes, that IL-3 may possibly be a factor responsible for T lymphocytes to be differentiating extra-thymically.

Animals↗
PubMed1993

Induction of specific tolerance by hepatic double-negative CD4-8- alpha beta T cells of mice immunized with allogeneic cells via the portal vein in vivo [corrected].

We immunized AKR/n (H-2k) spleen cells in BALB/c (H-2d) mice via the portal vein (pv) and investigated the role of hepatic mononuclear cells (MNC) in the induction of alloantigen-specific immune tolerance. MNC in the liver and spleen of pv-administered mice were demonstrated to abrogate the responses to AKR/n alloantigens in allogeneic MLR. On the contrary, MNC in the liver and spleen of mice administered subcutaneously with the same antigens showed greater responses than those of control mice. The tolerance induced by pv administration was alloantigen-specific and appeared earlier in hepatic MNC than in splenic MNC. Furthermore, hepatic MNC of pv-administered mice had a suppressive effect when these cells were added to allogeneic MLR, in which mitomycin C (MMC)-treated AKR/n splenic MNC were used as stimulator and control BALB/c splenic MNC were used as responder. Splenic MNC of pv-administered mice and hepatic MNC of control mice did not show such suppressive effects. Such suppression was alloantigen-specific, since no suppression was induced when hepatic MNC of pv-administered mice were added to a system using MMC-treated C57BL/6 (H-2b) splenic MNC. The alloantigen-specific suppression induced by hepatic MNC was abrogated by a depletion of TcR-alpha beta + cells but not of CD4+, CD8+, nor B220+ cells from hepatic MNC. These results suggested that alloantigen-specific suppressor cells appeared predominantly in the hepatic MNC of pv-administered mice and displayed the phenotype of TcR-alpha beta +CD4-8- double-negative T cells, although alloantigen-specific tolerance was induced in both hepatic and splenic MNC.

Animals↗
PubMed1993

Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.

The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor molecules are labeled with 40 nm-phi colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resolution of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor molecules obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were observed: (a) stationary mode, in which the microscopic diffusion coefficient is less than 4.6 x 10(-12) cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coefficient between 4.6 x 10(-12) and 1 x 10(-9) cm2/s) are confined within a limited area, probably by the membrane-associated cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diameter (0.04-0.24 microns2 in area), in which receptor molecules are confined in the time scale of 3-30 s, and that the long-range diffusion observed by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of molecules in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from approximately 60% to 8% (low- and high-calcium mediums, respectively).

Animals↗
PubMed1993

Age-associated increase of CD5+ B cells in the liver of autoimmune (NZB x NZW) F1 mice.

The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5+ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB x NZW) F1 mice. An age-associated increase of CD5+ B cells was demonstrated in the liver of these mice. Although CD5+ B cells (i.e., CD5+IgM+ and CD5+B220+) constituted a minor population of hepatic mononuclear cells (MNC) (< 5%) when mice were young (8 weeks), a large population of CD5+ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age-dependent increase of CD5+ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5+ B cells was the same as that of CD5+ B cells in the peritoneal cavity and spleen, showing dull-CD5, bright-IgM and dull-B220. High levels of CD5+ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5-day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM- and IgG-autoantibodies against double-stranded DNA and single-stranded DNA, despite the increased proportion of CD5+ B cells. On the other hand, peritoneal exudate cells produced only IgM-, but not IgG-, autoantibodies, whereas splenic cells were able to produce both IgM- and IgG-autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Age-dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice.

The liver is a major site of generation of extrathymic T cells with unique properties (e.g., expressing intermediate TCR and containing self-reactive clones). We investigated herein whether the levels of extrathymic alpha beta T cells varied in various organs as a function of age. A systematic examination of the number of mononuclear cells in various organs of BALB/c mice revealed that the number of hepatic MNC increased with age whereas the number of thymocytes decreased. These changes were more striking in mice fed under conventional conditions than under specific pathogen-free condition. The age-dependent changes in the number of mononuclear cells in the spleen and lymph nodes were minimal. Although the total proportion of alpha beta T cells in each organ remained constant, the staining patterns of TCR-alpha beta as shown by immunofluorescence profiles varied. The most prominent change was that intermediate TCR-alpha beta cells, which constituted a small population in the liver of young mice, expanded in the liver of older mice. Intermediate TCR cells appeared even in the periphery of older mice. These findings were confirmed by the appearance of extrathymic T cells with other unique properties, e.g., double-negative CD4-8- phenotype and CD44 expression. In athymic nude mice, only intermediate TCR cells were present in the liver and periphery. An age-dependent increase of intermediate TCR cells was also seen in these mice. Taken together with the result of bromodeoxyuridine-injection experiment, which showed an intensive in vivo proliferation of cells in the hepatic sinusoids, extrathymic T cells may differentiate predominantly in the liver and appeared even to the periphery in older mice.

Age Factors↗
PubMed1992

Estrogen administration activates extrathymic T cell differentiation in the liver.

In addition to T cell differentiation in the thymus, we have recently reported that extrathymic T cell differentiation occurs preferentially in the sinusoids of the liver. Although this extrathymic pathway is relatively minor in normal mice, it becomes predominant in mice with autoimmune diseases, athymic mice, and aged mice. In the present study, injection of normal male C3H/He mice, 6-8 wk of age, with 1 mg of estrogen resulted in an increase in mononuclear cells (MNC) yielded from the liver and a drastic decrease in thymocytes approximately 10 d after such injection. This unique modulation was not observed with hydrocortisone injection (5 mg/mouse, i.p.) nor with irradiation (5 Gy/mouse). Rather, these immunosuppressive treatments induced a simultaneous decrease in cell number in both the liver and thymus. A time-kinetics study on the cell number and spontaneous cell proliferation revealed that an increase in spontaneous cell proliferation in the liver preceded the increase in the number of liver MNC, and a decrease in spontaneous cell proliferation in the thymus preceded the decrease in the number of thymocytes. At this time, an enrichment of alpha/beta T cells with intermediate T cell receptors (TCRs), including forbidden T cell oligoclones and V beta 8+ cells, which are characterized as extrathymic alpha/beta T cells with unique properties, took place in the liver. On the other hand, the thymic atrophy induced by estrogen resulted in a prominent decrease in immature double-positive (CD(4+)8+) alpha/beta T cells with dull TCRs. These results indicate that estrogen administration activates an extrathymic pathway of T cell differentiation in the liver and reciprocally inactivates the intrathymic pathway. As extrathymic T cells have unique characteristics such as autoreactivity, the present findings might be intimately related to a female predominance of autoimmune diseases and suggest a possible role of estrogen in this phenomenon.

Animals↗
PubMed1992

Details of an isolation method for hepatic lymphocytes in mice.

The liver comprises a unique lymphocyte population, i.e., extrathymic alpha beta T cells with TcR of intermediate intensity. In the present study, we attempted to determine what pretreatments were appropriate to isolate hepatic mononuclear cells (MNC) containing such intermediate alpha beta TcR cells in mice. Hepatic MNC were isolated from untreated mice and mice subjected to either bleeding or liver perfusion, and the intermediate alpha beta TcR cells in each preparation were identified. For reasons of simplicity, cell purity and cell yields, hepatic lymphocytes should be obtained from mice subjected to total bleeding. Additional information on extrathymic alpha beta T cells obtained by using the recommended method is also presented.

Animals↗
PubMed1992

Activation of extrathymic T cells in the liver and reciprocal inactivation of intrathymic T cells by bacterial stimulation.

We recently demonstrated that the liver might be a major site of extrathymic T cell differentiation, including both alpha beta and gamma delta T cells. This extrathymic pathway in the liver, which has a relatively minor role in normal young mice, is activated in mice under bacterial stimulation. In the present study, we investigated how the extrathymic and intrathymic T cell differentiations were mutually related in mice injected intravenously with 10(8) heat-killed Escherichia coli. Three days after stimulation, extrathymic T cells in the liver were observed to be prominently activated in terms of increases in the total number of cells yielded, spontaneous cell proliferation in in vitro culture, and intermediate alpha beta TCR cells. Intermediate alpha beta TCR cells were extrathymic T cells uniquely seen in the liver. However, at the same time intrathymic T cells were profoundly inactivated, showing decreases in the number of thymocytes (more than 90% atrophy), spontaneous cell proliferation, and dull TCR cells with double positive CD4+8+ phenotype. With time, these responses were reversed and normal states were regained. These results suggested that extrathymic and intrathymic T cells are always activated or inactivated in the opposite direction, and that the liver and the thymus are dynamic immune organs. It raises the possibility that the extrathymic T cell differentiation in the liver and the intrathymic T cell differentiation may be reciprocally regulated by certain factors.

Animals↗
PubMed1992

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method.

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.

Animals↗
PubMed1992

Lymphotoxin activates hepatic T cells and simultaneously induces profound thymic atrophy.

We have recently demonstrated that the liver may be a major site of extrathymic T-cell differentiation. This hepatic pathway was shown to be activated in mice injected with heat-killed bacteria. It is conceivable that the resulting activation of macrophages or lymphocytes, and the production of cytokines may be responsible for a subsequent activation of hepatic T cells. In this context, we investigated the possibility of whether certain cytokines may activate hepatic T cells. It was demonstrated that the administration of lymphotoxin [tumour necrosis factor-beta (TNF-beta)] more than doubled the number of hepatic mononuclear cells (MNC) yielded 3-5 days after the treatment. More strikingly, such treatment induced profound thymic atrophy and resulted in a decrease of more than 95% in the number of thymocytes. Spontaneous proliferation in an in vitro culture of hepatic MNC from treated mice increased, and inversely such activity of thymocytes decreased. The increased number of hepatic MNC was mainly due to an increase in intermediate alpha beta T-cell receptor (TcR) cells, which are extrathymic T cells uniquely seen in the liver. On the other hand, the thymic atrophy was caused by the prompt apoptotic death of dull alpha beta TcR cells with double-positive (DP) CD4+ CD8+ phenotype. These results indicate that lymphotoxin may be one of the factors that activates extrathymic T cells in the liver and at the same time inhibits intrathymic T-cell differentiation.

Animals↗
PubMed1991

Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes: a pulse ESR spin labeling study.

Transport and diffusion of molecular oxygen in phosphatidylcholine (PC)-cholesterol membranes and their molecular mechanism were investigated. A special attention was paid to the molecular interaction involving unsaturated alkyl chains and cholesterol. Oxygen transport was evaluated by monitoring the bimolecular collision rate of molecular oxygen and the lipid-type spin labels, tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid. The collision rate was determined by measuring the spin-lattice relaxation times (T1's) in the presence and absence of molecular oxygen with long-pulse saturation-recovery ESR techniques. In the absence of cholesterol, incorporation of either a cis or trans double bond at the C9-C10 position of the alkyl chain decreases oxygen transport at all locations in the membrane. The activation energy for the translational diffusion of molecular oxygen in the absence of cholesterol is 3.7-6.5 kcal/mol, which is comparable to the activation energy theoretically estimated for kink migration or C-C bond rotation of alkyl chains [Träuble, H. (1971) J. Membr. Biol. 4, 193-208; Pace, R. J., & Chan, S. I. (1982) J. Chem. Phys. 76, 4241-4247]. Intercalation of cholesterol in saturated PC membranes reduces oxygen transport in the headgroup region and the hydrophobic region near the membrane surface but little affects the transport in the central part of the bilayer. In unsaturated PC membranes, intercalation of cholesterol also reduces oxygen transport in and near the headgroup regions. In contrast, it increases oxygen transport in the middle of the bilayer. On the basis of these observations, a model for the mechanism of oxygen transport in the membrane is proposed in which oxygen molecules reside in vacant pockets created by gauche-trans isomerization of alkyl chains and the structural nonconformability of neighboring lipids, unsaturated PC and cholesterol in particular, and oxygen molecules jump from one pocket to the adjacent one or move along with the movement of the pocket itself. The presence of cholesterol decreases oxygen permeability across the membrane in all membranes used in this work in spite of the increase in oxygen transport in the central part of unsaturated PC-cholesterol membranes because cholesterol decreases oxygen transport in and near the headgroup regions, where the major barriers for oxygen permeability are located. Oxygen gradients across the membranes of the cells and the mitochondria are evaluated. Arguments are advanced that oxygen permeation across the protein-rich mitochondrial membranes can be a rate-limiting step for oxygen consumption under hypoxic conditions in vivo.

Animals↗
PubMed1991

Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells.

A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.

Animals↗
PubMed1991

Influence of phospholipid unsaturation on the cholesterol distribution in membranes.

Over the last half decade, we have studied saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes, with special attention paid to fluid-phase immiscibility in cis-unsaturated PC-cholesterol membranes. The investigations were carried out with fatty acid and sterol analogue spin labels for which reorientational diffusion of the nitroxide was measured using conventional ESR technique. We also used saturation recovery ESR technique where dual probes were utilized. Bimolecular collision rates between a membrane-soluble square-planar copper complex,3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato)copper(II) (CuKTMS2) and one of several nitroxide radical lipid-type spin labels were determined by measuring the nitroxide spin-lattice relaxation time (T1). The results obtained in all these studies can be explained if the following model is assumed: 1) at physiological temperatures, fluid-phase micro-immiscibility takes place in cis-unsaturated PC-cholesterol membranes, which induces cholesterol-rich domains in the membrane due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the cis double bond of the alkyl chains of unsaturated PC. 2) The cholesterol-rich domains are small and/or of short lifetime (10(-9) s to less than 10(-7) s). Our results also suggest that the extra space that is available for conformational disorder and accommodation of small molecules is created in the central part of the bilayer by intercalation of cholesterol in cis-unsaturated PC membrane due to the mismatch in the hydrophobic length and nonconformability between cis-unsaturated PC alkyl chains and the bulky tetracyclic ring of cholesterol.

Cholesterol↗