LDL transcytosis by protein membrane diffusion

The International Journal of Biochemistry & Cell Biology 36 (2004) 519–534

LDL transcytosis by protein membrane diffusion夽 Elena S. Kuzmenko1 , Siamak Djafarzadeh2 , Z. Petek Çakar3 , Klaus Fiedler∗ Division of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel CH-4056, Switzerland Received 1 July 2003; received in revised form 18 August 2003; accepted 1 September 2003

Abstract Endothelial cell (EC) cultures of different, selected vascular beds and/or organs were screened for receptor-mediated transport of proteins with a semipermeable filter assay. In SVEC4–10 cells, a mouse lymphoid endothelial cell line, orosomucoid, albumin, insulin and LDL were transcytosed from the apical (luminal) to basal (abluminal) side by a receptor-mediated pathway. Specific LDL transcytosis involved transport of intact LDL. A pathway of degradation of LDL and basal release involved vesicles in transport to lysosomes and amino acid merocrine secretion. This newly described transcellular passage of LDL via lysosomes, as well as the standard pathway, were reduced to 70% by PEG(50)–cholesterol (PEG–Chol). Combined results of temperature-dependence analysis and PEG(50)–cholesterol sensitivity show that two pathways contribute to general LDL transcellular passage. We suggest a mechanism of domain hopping by protein membrane diffusion of receptors as the pathway for intact LDL delivery. Based on theoretical considerations we propose that active transport by protein membrane diffusion can be facilitated by an organizational structure of lipid microdomains and polar cellular organization. © 2003 Elsevier Ltd. All rights reserved. Keywords: Endothelia; Diffusion; Transcellular; Raft; Molecular shuttle

1. Introduction Endothelial exchange of molecules across the capillary cells is by a paracellular pathway or transcellular route (Dvorak & Feng, 2001; Kellen & 夽

This work was presented at the EURESCO/EMBO Conference “Microdomains, Lipid Rafts and Caveolae”, Tomar, Portugal, 17–22 May 2003. ∗ Corresponding author. Tel.: +41-61-321-1395; fax: +41-61-321-1395. E-mail address: k [email protected] (K. Fiedler). 1 Present address: Pediatric Surgery, Kantonssspital Basel, Basel CH-4031, Switzerland. 2 Present address: Institut für Biochemie und Molekularbiologie, Universität Bern, Bern CH-3012, Switzerland. 3 Present address: Department of Molecular Biology and Genetics, Istanbul Technical University, T-80626 Maslak, Turkey.

Bassingthwaighte, 2003; Michel & Curry, 1999; Parton, 2003; Simionescu & Simionescu, 1991). The transcellular pathway regulates selective nutrient supply to tissues and organs and has been found to involve clathrin-coated and plasmalemmal vesicles, transendothelial channels and/or vesiculo-vacuolar organelles, or transporters and membrane channels. It was analyzed for low-density lipoprotein (LDL) by morphology in rat lung (Vasile, Simionescu, & Simionescu, 1983) and in a rabbit culture system of aortic endothelial cells (EC; Hough, Ross, Navab, & Fogelman, 1990). In lung, the suggestion was put forward to allow for partial transfer of cholesterol to lung parenchymal cells without the apoprotein moiety, as well as transport of intact LDL, in a description of the intracellular transport process (Nistor & Simionescu, 1986). Contrary to these observations, in

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brain microvascular endothelial cells in tissue-culture, the majority of LDL was transcytosed as intact LDL particle (Dehouck et al., 1997). The polarity of epithelial cells is well established (Mostov, Su, & Ter Beest, 2003; Simons & van Meer, 1988), but endothelial polarity has as yet not been described in detail (Stolz & Jacobson, 1992; Uehara & Miyoshi, 1999) and is of pivotal role to carry out vectorial functions. Several markers were found to be transcytosed in a receptor-mediated fashion, such as amyloid-␤ mediated by RAGE (receptor for advanced glycation endproducts) (Deane et al., 2003; Milici, Watrous, Stukenbrok, & Palade, 1987; Minshall et al., 2000; Niles & Malik, 1999; Predescu, Predescu, McQuistan, & Palade, 1998; Schnitzer, Oh, Pinney, & Allard, 1994; Simionescu & Simionescu, 1991). Amyloid-␤ accumulates in the brain by this process and causes endothelin-1 production (Deane et al., 2003). In continuous endothelia, plasmalemmal vesicles have been shown morphologically to be involved in transcytosis and have been equated with caveolae (Milici et al., 1987; Palade, 1953; Palade & Bruns, 1968). The findings in microvascular muscle capillaries established that tracer density on the tissue side of junctions was less than in the interstitium in pulse experiments (Simionescu, Simionescu, & Palade, 1975). This suggests, that exchange occurs by transcellular transport. New results however demonstrate that caveolin-1, a major coat protein of caveolae, is not essential for the viability of −/− gene-deleted mice (Drab et al., 2001; Razani et al., 2001), suggesting that the representation about transendothelial transport must be adjusted, and a compensatory mechanism, the opening of endothelial junctions, exists to allow paracellular transport. For viruses and bacteria the uptake by caveolae is a process which is induced and involves signaling mechanisms and a novel compartment, the caveosome (Pelkmans, Puntener, & Helenius, 2002). Thus, it may represent a feature of caveolae that can be exploited in pathogen invasion. We resorted to temperature-shift experiments in our transport analysis and used the GM1-pathway inhibitor PEG(50)–cholesterol (PEG–Chol; Baba et al., 2001; Ishiwata, Sato, Vertut-Doi, Hamashima, & Miyajima, 1997). GM1 has been found concentrated in caveolae by electron microscopy (Parton, 1994), but is rather evenly distributed at the plasma membrane and excluded from clathrin-coated struc-

tures (Nichols, 2003). We have here characterized the endothelial cell line SVEC4–10 for transcytosis analysis. It is derived from SV40 strain 4A infected mice and isolated from the axillary lymph node (O’Connell & Edidin, 1990), thus it may be of blood vascular or lymph vascular origin. The paucity of markers has not yet allowed to differentiate these possiblities (Podgrabinska et al., 2002), however, both cell types generally were characterized for expression of von Willebrand factor (Djoneidi & Brodt, 1991; Borron & Hay, 1994). The concept that we propose explains molecular details of a shuttle mechanism. 2. Methods 2.1. Materials Inulin-carboxyl[14 C] was from ICN. Cell culture media and fetal calf serum (FCS) were from Gibco BRL and collagen (rat tail, type I) was purchased from Collaborative Biomedical Products. Trans-endothelial electrical resistance (TEER) was measured with a Millicell electrode from Millipore. ZO-1 antibody was from Zymed Laboratories. Cy2-conjugated goat anti-rabbit IgG antibody was purchased from Jackson Immuno Research Laboratories. Texas-Red albumin was from Molecular Probes. Insulin was from Calbiochem and orosomucoid from Sigma. PEG(50)–cholesterol was a kind gift of T. Baba. 2.2. Cell lines and screen ECV304, FBHE, CPAE and SVEC4–10 cells were obtained from the American Type Culture Collection and cultured and passaged according to the recommendations of the cell bank. SVEC4–10 cells were cultured in DMEM (Eagle), 10% or 3% fetal calf serum or lipoprotein-deficient serum (LPDS) and used from passage number 32–52. EA·hy926 cells were a kind gift from Cora-Jean Edgell (Chapel Hill, USA) (Edgell, McDonald, & Graham, 1983). For TEER measurements, cells were plated on filter inserts (NUNC 0.4 ␮m, 25 mm diameter) and measured in the respective growth medium. Inserts were coated on the upper side with collagen. Radioactive inulin was added to the growth medium of the upper compartment

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(on day 4) for permeability studies and sampled from the lower compartment after 1 h. 10 mM EGTA addition was carried out with a medium containing DMEM, 10% FCS and 20 mM Hepes pH 7.4. The experiment was conducted after equilibration of cells and media at rt in the absence of CO2 . For immunofluoresence analysis SVEC4–10 cells were grown for 3 days as monolayers as described above using 10% FCS/DMEM. Antibody incubation was carried out by standard filter procedures (Fiedler, Lafont, Parton, & Simons, 1995) with PBS (containing 0.5 mM CaCl2 , 0.5 mM MgCl2 ) for washes, 4% paraformaldehyde with 0.1 mM CaCl2 and 0.1 mM MgCl2 in PBS as fixative, and permeabilization with 0.2% Triton-X 100 in PBS for 6 min. ZO-1 and Cy2-conjugated anti-rabbit IgG antibody were used at a dilution of 1:80. Samples were observed with a Leica DM R confocal microscope with an APO 100× oil immersion objective. 2.3. LDL purification from blood LDL (density 1.019–1.063 g/ml) was isolated from the plasma of healthy donors using sequential buoyant density centrifugation techniques, with the use of potassium bromide for density adjustments (Havel, Eder, & Bragdon, 1955). LDL was exhaustively dialyzed against 0.15 M NaCl before Lowry determination of protein concentration and sterilization by filtration through 0.2 ␮m Gelman filters. EDTA (1 mM) and butylated hydroxytoluene (0.1 mM) were present during all isolation and dialysis procedures. SDS-PAGE revealed that LDL preparations contained only apo B-100 protein and were free of protein fragments generated during LDL oxidation. LDL was 125 I-labeled on tyrosine residues using the iodine monochloride method (Goldstein, Basu, & Brown, 1983). LPDS was prepared from FCS by ultracentrifugation at the density of 1.25 g/ml adjusted with solid NaBr. 2.4. Transcytosis assay The passage of LDL through SVEC4–10 monolayers was studied using a two-chamber culture system. Endothelial cells were plated on the upper side of the filters in 3.6 ml of DMEM with 10% FCS with

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a density of 5 × 105 –7 × 105 cells per filter. Cells were cultured for 3 days. The transcytosis experiment was carried out by washing the EC with DMEM containing 2 mg/ml BSA, filters were transferred to a six-well plate containing 2 ml of serum free medium DMEM/BSA (lower compartment). At time zero, a known amount of 125 I-LDL (usually 30 ␮g protein/ml; as indicated in the figure legends) in DMEM/BSA was added to the upper compartment (1.5 ml). Media levels in the two compartments were adjusted to the same level and cells were kept at 37 ◦ C. At several time points, filters were transferred to the next well. 1 ml aliquots were collected from the lower compartments and precipitated with trichloroacetic acid. Soluble and insoluble fractions were counted in a ␥-counter. Transcytosis was examined both in the absence and in the presence of 25/30-fold excess of unlabeled LDL. Incubations were carried out at 37 ◦ C or 4 ◦ C. For LPDS treatment cells were grown for 48 h. Two experimental sets were analyzed and pooled confidence intervals (CIs) are given. PEG–Chol preincubation was carried out by adding 0.5–10 ␮M PEG–Chol from a stock solution (dissolved in H2 O) to DMEM with or without BSA. BSA was not found to significantly affect the 30 min preincubation and experimental result. 2.5. Statistics Confidence intervals (α = 0.5) for specific transcytosis and transport/degradation were calculated conventionally (see Wilks, 1948) for 4d.f. and a tα of 0.741 (for small sample size) by programming on a computer. The confidence limit of 50% was chosen since the standard deviation (s) (for total and non-specific transcytosis and transport/degradation) relates to α = 0.68. The equation used was (X denotes the sample mean):
 1 ((n − 1)s2 + (n − 1)s 2 ) 1 ¯ ¯ X − X ± tα + n+n −2 n n The statistical significance was estimated in a one-sided t-like test each determining the tail area probability since the χ2 -test is not recommended. The control condition was measured routinely and to combine experiments conducted on different days. Pooled confidence intervals are given as implied by Wilks (1948), i.e. calculated for n = 3 versus n = 3

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filters for the same experimental set and combined as average from a total experimental series. 2.6. ESEM For environmental scanning EM (Donald, 2003), cells were grown on collagen-coated filter inserts, washed and fixed as described above for immunofluorescence experiments, and then excised and mounted. The chamber was adjusted in humidity (at rt), scans were performed intermittently to obtain scans of humidified samples.

3. Transcytosis screen We screened for receptor-mediated transcytosis in stable polarized endothelial and epithelial cells. Endo-epithelial cells were also included in this screen, since frequently cell fusion facilitates survival of primary cultures of endothelia and in this way a model system of heterokaryons is generated. The epithelial cell line (ECV304) was assumed to be endothelial at the beginning of this study. Cells were cultured on collagen-coated polycarbonate filters to assay for monolayer growth. Out of twelve different cultured cells, five were growing as monolayers and showed trans-endothelial electrical resistance (Fig. 1a). Fetal bovine heart endothelial (FBHE) and calf pulmonary artery endothelial (CPAE) cells displayed a TEER of 24 and 54 ·cm2 , respectively, at day 4 after plating on filters. Both, FBHE and CPAE cells are primary cultures of endothelial cells derived from microvasculature and large vessels, respectively. SVEC4–10 cells derived from mice axillary lymph nodes (O’Connell & Edidin, 1990) had a high TEER of 105 ·cm2 at day 4. These cells were found suitable for monolayer studies and were further used. The TEER was correlated with the flux of the inert marker inulin across the cell layer to test for the integrity of junctions (data not shown). SVEC4–10 cells provided the largest barrier to inulin permeation followed by CPAE, EA·hy926, ECV304 and FBHE cells. The TEER of SVEC4–10 cells rose from 30 ·cm2 at day 1 after plating to 137 ·cm2 at day 4 and inulin permeation increased linearly within the time of 4 h at this day, suggesting that the junctions in test conditions remained intact (i.e. sampling of medium

Fig. 1. Comparison of cells. Electrical resistances (a) of cell lines (SVEC4–10, EA·hy926, ECV304) and primary cultures of cells (FBHE, CPAE). Error bars for n = 2. Electrical resistance (b) and junctional labeling (ZO-1) (c) of mouse endothelial SVEC4–10.

and transcytosis assay). Short incubation of the cells with EGTA at constant pH lead to a rapid decrease in TEER which slowly recovered upon EGTA removal (Fig. 1b). This suggests that endothelial SVEC4–10 cells have closed calcium-dependent junctions. The majority of junctions were well organized in this cell line (as visualized by ZO-1 labeling) (Fig. 1c). EA hy926 cells displayed a TEER of 70 ·cm2 at day 4

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Fig. 2. Analysis of LDL transcytosis and degradation. Transport from the luminal to the abluminal side was measured by sampling the basal compartment and quantified as acid-insoluble (a) and acid-soluble (b) LDL radioactivity. Cells in (c) and (d) were incubated at 37 ◦ C (standard conditions) and 4 ◦ C during the transport assay. Error bars denote standard deviations for the total and non-specific transport (for n = 3; n = 3). 50% confidence intervals (CIs) are indicated by bars in all panels for the specific transport (small sample size).

LDL Transcytosis ng/ml

(a)

15

total

10

nonspecific specific

5

0 0

60

120 time, min

180

LDL Degradation and Transport ng/ml

(b) 800

total nonspecific

600

400

200

specific 0

Specific LDL Transcytosis ng/ml

0

(c)

60

120 time, min

180

37˚C 4

2

4˚C

0 0

Specific LDL Degradation and Transport ng/ml

after plating. They are heterokaryons of human umbilical vein endothelial cells (HUVEC) and epithelial A549 cells (Edgell et al., 1983). ECV304 cells, transformed human cells of epithelial origin (Dirks et al., 1999; Takahashi, Sawasaki, Hata, Mukai, & Goto, 1990), showed a TEER of 14 ·cm2 . Since SVEC4–10 cells were the only transformed stable endothelial line that remained in this analysis, we screened them for receptor-mediated transport of albumin, insulin, orosomucoid (␣-acid glycoprotein) and LDL. These molecules were earlier shown to be exchanged in various vascular beds (Milici et al., 1987; Minshall et al., 2000; Predescu et al., 1998; Simionescu & Simionescu, 1991) but were not analyzed in detail in one cell line. Tracers were added to SVEC4–10 cells and receptor-mediated transport was analyzed. LDL was of the highest purity in intact non-oxidized form and no degradation of purified or sham (i.e. collagen-coated filter) incubated LDL could be shown by SDS-PAGE. LDL transport was analyzed for transcytosis of intact and en route degraded LDL by sampling medium from the abluminal compartment over a time of 3 h followed by analysis of acid-insoluble (Fig. 2a) and acid-soluble radioactivity (Fig. 2b) (Goldstein et al., 1983). LDL transport was 14.1 ng/ml in total (Fig. 2a) and included 8.1 ng/ml non-specific and 6.0 ng/ml specific receptor-mediated transcytosed material within 3 h (comparable to 4 ng/h specific transcytosis/4.5 cm2 filter). This value is reduced relative to the approximately 40 ng/h and 4.5 cm2 filter transport for co-cultures of brain capillary endothelial cells and astrocytes (Dehouck et al., 1997) and suggests fundamental differences in LDL transport and/or metabolism in SVEC4–10 cells. Receptor-mediated LDL degradation and release on the basal side (Fig. 2b) was approximately 10-fold higher than the specific transport of acid-insoluble LDL (Fig. 2a) and distinguishes SVEC4–10 cells of

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(d)

60

120 time, min

180

200

37˚C

100

4˚C 0 0

60

120 time, min

180

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lymph node origin from brain microvascular endothelial cells which do not degrade LDL in transcytosis (Dehouck et al., 1997). Largest amounts of LDL were transcytosed, the other tracers were transported in a receptor-mediated fashion in smaller quantities. The saturation behaviour of receptor-mediated intact LDL transcytosis was not unimodal (data not shown).

4. Transcellular transport in SVEC4–10 The different transcellular pathways in endothelia have not been elucidated in detail on the molecular level but include caveolae that bud in a dynamin one-dependent way (Schnitzer, Oh, & McIntosh, 1996; Oh, McIntosh, & Schnitzer, 1998) as well as clathrincoated vesicles (Nistor & Simionescu, 1986; Vasile et al., 1983). Plasmalemmal vesicles have been defined by their featureless cytoplasmic aspect and characteristic neck structure, different from clathrin-coated vesicles and visible in the electron microscope, but not always resolved. Reagents to selectively perturb a general third pathway of endocytosis for monolayer transcytosis studies, utilized by many toxins, have not been available (Sandvig & van Deurs, 2002). To analyze the pathways we therefore first resorted to temperature-shift experiments previously used for studies of clathrin-mediated uptake (Schmid & Smythe, 1991). LDL degradation and release was reduced by a factor five at 4 ◦ C relative to 37 ◦ C (Fig. 2d) but receptor-mediated transcytosis of acid-insoluble LDL showed only a two-fold temperature-dependent reduction (Fig. 2c). The temperature-dependence of

LDL degradation and basal release is inline with the analysis of clathrin-mediated uptake in semiintact cells and since endothelial transcytosis may include lysosomal intermediates (Vasile et al., 1983) we conclude that the indirect pathway is via lysosomes as intermediate station. However, the two-fold temperature dependence of the specific direct, intact LDL pathway suggests that this route follows a thermodynamically different pathway. Secondly, since it has been shown that LDL internalization to lysosomes involves the ApoB, E-receptor (Goldstein, Basu, Brunschede, & Brown, 1976) SVEC4–10 cells were preincubated with lipoprotein-deficient serum (LPDS) for 48 h prior to the experiment to test for its involvement (Table 1). This receptor is involved in uptake of LDL and chylomicrons. The treatment was shown to upregulate LDL receptor expression in endothelia (Antohe, Poznansky, & Simionescu, 1999) and did not decrease TEER (data not shown). Moreover, the caveolin expression levels were not altered in LPDS (data not shown) and we infer that the cells are stationary (Galbiati et al., 2001) and likely that the number of caveolae and presumably caveolae-mediated flow volume was not elevated. LDL degradation and release on the abluminal side was increased 1.8-fold (3 h time point; P = 0.005) after LPDS treatment suggesting that the LDL receptor mediates lysosomal uptake. Total release of monoiodotyrosine was increased 2.3-fold (data not shown). In contrast, transcytosis of acid-insoluble LDL was increased 1.3-fold (3 h time point; P = 0.02) (Table 1) implying that a different receptor is involved in the direct route.

Table 1 LDL transport in SVEC4–10 cells Growth medium

Transport (h)

Specific transcytosed 125 I-LDL (ng/ml)

Range (ng/ml)

FCS

1 2 3

1.6 3.5† 5.1∗

1.0–2.4 2.8–4.5 3.6–6.6

LPDS

1 2 3

1.7 3.9 6.7†

0.8–2.3 2.2–6.8 4.0–13.8

The confidence coefficient α was 0.5. Confidence interval (CI) one-sided. ∗ P ≤ 0.05. † P ≤ 0.1.

CI (%)

Specific degraded/transported 125 I-LDL (ng/ml)

Range (ng/ml)

CI (%)

7.0 4.7 7.7

36.5 74.6∗ 106.0∗

13–63 36–111 63–154

20.4 13.4 9.2

8.0 10.1 10.7

43.0 117.0∗ 188.7∗

26–56 99–143 155–224

13.6 8.4 6.1

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(a)

10 0.05

9

Specific LDL Transcytosis ng/ml

525

8 7

0.05

6

0.04

5

Control PEG-Chol

0.06

0.06

4 3

0.25

2 1 0 1

2

3

Specific LDL Degradation and Transport ng/ml

time, hours (b)

180 0.008

160 140

0.04

0.04

120 100

Control PEG-Chol

0.06

80 0.08

60 0.1

40 20 0 1

2

3

time, hours Fig. 3. PEG–Chol effect on LDL transport: (a) specific transcytosis, acid-insoluble; (b) specific transcytosis, acid-soluble. Pooled confidence intervals (CIs) of n = 3 vs. n = 3 filters of individual experiments averaged are indicated, one-sided, by bars. The confidence coefficient α was 0.5.

Thirdly, we analyzed whether in our transport assay transport could be inhibited by select pharmacological treatment of the bilayer with PEG(50)–cholesterol. Treatment of the bilayer with cyclodextrin analogues is not applicable to monolayer cultures. PEG(50)–cholesterol is a poly (ethylene glycol)-modified (n = 50) cholesterol molecule. This reagent was shown to inhibit GM1 uptake selectively but does not affect the clathrin-mediated pathway (Baba et al., 2001; Ishiwata et al., 1997). The results show that the transcytotic exchange of both acid-insoluble (Fig. 3a) and acid-soluble LDL (Fig. 3b) is reduced by 30% with a P = 0.04 for

both pathways (3/1 and 2 h time point, respectively; condition arbitrarily chosen) suggesting that this fraction of transport is related to the GM1 route. Analysis of the raw values shows a systematic error. Its correction would increase the significance of the results. This implicates multiple routes in the direct and indirect pathway since receptor-mediated transcytosis was analyzed in our experiments, PEG(50)– cholesterol was shown to inhibit uptake of GM1 (Baba et al., 2001) and, especially, since the temperature dependence of the direct pathway is unlikely to be consistent with a vesicular transport process.

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5. Scanning EM

6. Membrane traffic in endothelia

We tested for the presence of pores in the cell layer by scanning SVEC4–10 cells in the environmental scanning electron microscope (ESEM) (Donald, 2003). Based on fluid exchange in muscle (Michel & Curry, 1999) one expects approximately 20,000 transported vesicles per endothelial cell and minute corresponding to 10 exchanges/␮m2 within equivalent time, but in this scenario one postulates membrane lipid diffusion in reverse transport to account for vectorial uptake. Instead, we find by scanning EM that SVEC4–10 cells contain numerous pores that are visible as darkened areas within the cell perimeter (Fig. 4). At this maximal resolution, the areas seem to contain clusters of pores. It may thus be general, that fluid exchange occurs via pores in endothelia (Frokjaer-Jensen, 1980; Simionescu & Simionescu, 1991).

6.1. Receptor-mediated transcytotic domain hop The relationship of transendothelial channels to vesiculo-vacuolar organelles, which provide conduits for transvascular exchange, remains to be determined and we refer to them as pores in a further discussion (Clough, 1991; Dvorak & Feng, 2001; Feng et al., 1997; Frokjaer-Jensen, 1980; Simionescu & Simionescu, 1991; Van Deurs, Roepstorff, Hommelgaard, & Sandvig, 2003). For microvascular endothelia we consider the possibility that pores are also constituted of fenestrae or gaps; the distinction of clefts and pores of different sizes has recently been further studied (Kellen & Bassingthwaighte, 2003). Contrary to our expectations we find the two-fold reduction of the intact receptor-mediated LDL transcytosis

Fig. 4. ESEM. Scan of SVEC4–10 cells. Darkened areas (approximately 500 nm in diameter each) that may correspond to pores or clusters of pores are labelled with an asterik. Further similar areas are visible but not reproduced at high enough quality.

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pathway at 4 ◦ C relative to 37 ◦ C. The receptors for this pathway remain to be analyzed and could include members of the LDL-receptor family or novel proteins (Antohe et al., 1999; Hajjar & Haberland, 1997; Rudenko et al., 2002; Willnow, Nykjaer, & Herz, 1999), but, most likely, not the ApoB, E-receptor. The low temperature-dependence was, to our knowledge, hitherto unknown and we conclude that remarkable features of its enzymology and/or unexpected bilayer fluidity and pores allow this traffic to proceed to this rate at 4 ◦ C (Fig. 5). The cellular membrane bilayer is in a tension-free state, neglecting vesicles, and usually no phase transitions occur within this temperature range. The plasma membrane is not permeable to substances at low temperature, with strong fluctuations in shape close to temperature-induced transitions only (Heimburg, 2000), suggesting that flexibility of the bilayer is warranted at 4 ◦ C. The physical chemistry of membrane bilayer fluidity determination is associated with problems (Hare, Amiell, & Lussan, 1979). However, is the mobility of docosahexaenoyl acyl chains not significantly affected by lowering the temperature (Binder & Gawrisch, 2001) and the ordering tensor of mixtures of DPPC and cholesterol decreases 30% at 37 ◦ C relative to 4 ◦ C (Lou, Ge, & Freed, 2001). If representative for other unsaturated lipids and mixtures, this speaks for its high fluidity. Moreover, for some mixtures of lipids the ultrasonic velocity varies only little with temperature (Hianik et al., 1998; Schrader et al., 2002). In model calculations, the temperature effect on the diffusion coefficient of membrane objects is primarily dependent on the changes of viscosity of the bilayer (Kamm, 2002; Saffman & Delbruck, 1975), which is influenced and/or measured by above parameters (neglecting the shear viscosity for the latter; see also (Herzfeld & Litovitz, 1959)), and thus the mean square displacement is expected to be high even at 4 ◦ C. One would predict large hydrophobic domains to interact in this transport process if mediated by vesicles, which cannot be excluded, or inverse temperature dependence may occur depending on the specific type of interaction (Lipschultz, Yee, Mohan, Li, & Smith-Gill, 2002). This would leave protein interactions unthrottled at low temperature. Alternatively, a domain hop of a receptor from luminal to abluminal via pores, as suggested above, provides the mechanism to deliver the cargo to its destination

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abluminally. This could be driven along channels that only have a transient connection for particle flow or receptor-mediated transport. This distinguishes a gated pore (vesiculo-vacuolar organelle) (Fig. 5e) from a transendothelial channel (Fig. 5a–d) but is implicit to the model. Domain hopping, which in its original description refers to membrane protein diffusion in erythrocytes (Kucik, Elson, & Sheetz, 1999; Simson et al., 1998; Tomishige & Kusumi, 1999; Tsuji & Ohnishi, 1986) alternating between confined (corralled) diffusion in one domain, hop, and confined (corralled) diffusion in another, would contribute to a process that, depending on the junctions or availability of pores could lead to directional transport across the cell layer. It is not implied that junctions in the form of a corral border exist but the cytoskeletal organization of endothelia could nevertheless contribute to gating, and tensegrity (tensional integrity) (Wang et al., 2001) of the cell layer could signal across from one domain to the other to allow for rapid protein-mediated membrane fusion (Fig. 5e). Alternatively, the cortical cytoskeletal barrier could be modulated and removed, and this would transform racemose or grape-like structures of vesicles (Anderson, 1998) into shapes of different surface. This process may not require membrane fission but only fusion in that the levy in lipid surface could implicate channel conformal diffusion and shape fluctuations (Lipowsky, 1995; Seifert, 1997), and translation of bilayer leaflets in a rapid mechanism, providing different lipid composition and/or lipid area enlargement at the site of fusion (Fig. 5e). Fusion would be followed by fission and, furthermore, a receptor-mediated gating could enhance specificity of transport. If, however, vesicles were involved in transcellular transfer, one could predict, that they have to overcome a large kinetic barrier due to the actin cortical organization (Lang et al., 2000). In the other scenario of protein membrane diffusion which we consider the most likely, the membrane skeleton attached to membrane proteins, would provide a flexible barrier to protein diffusion. Proteins could interact with the barrier, just as with lipids, diffuse freely, and may pass the barrier due to thermal undulations (Brown, 2003; Marsh & Horvath, 1998; Saxton & Jacobson, 1997). This step remains unexplored in endothelial transcellular transport. The binding constants of ligands to hypothetical receptors in directional transport in endothelia, just

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Fig. 5. Model of hop diffusion, simplified: (a) no receptor, ligand; (b) receptor, ligand; (c) receptor, extracellular co-receptor, ligand. Ligand binding to ECM will provide a sink. The apparent affinity of the receptor to the ligand is higher in (c), due to higher effective concentration on the ECM side. (d) Ratchet model: Receptor, intracellular/membrane tether, ligand or (d) flow model: Receptor, ligand, flow or (d) combined ratchet-flow model. The affinities of receptors to intracellular tethers and/or lipids involve the ratchet, thus, cellular organization delimits the starting points of walk for receptors (mechanical anisotropy), and introduces point-spread of released ligand–receptor pairs, and flow will impose viscous forces on bound ligands. (e) Vesiculo-vacuolar organelle, ligand, cytoskeletal communication. Membrane fusion of attached abluminal with luminal vesicular structures is triggered and allows transient opening of a transendothelial pore. (a–e) The kinetics of blood flow (up into the stochastic regime) will affect viscous forces, reduce ligand amount in the boundary layer, and may alternate between influx and backflux to the adjacent layer through transient connections and/or transendothelial channels.

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as for many other directional processes, remain to be defined. A receptor expression in specific organs or vascular beds could provide a sink for organand tissue-selective delivery and facilitate exchange by transverse transport in that organ (Fig. 5b). We have found that heparin incubation in the presence of LDL will decrease LDL internalization less than incubation with excess, non-labelled LDL (data not shown) (Goldstein et al., 1976). This suggests that glycocalyx providing a sink on endothelial cells, just as on fibroblasts, is of lower affinity than the LDL receptor(s). Thus, the glycocalyx will elevate the local concentration of ligand on the luminal side and possibly provide release abluminally to glycocalyx due to mass action and subendothelial retention (Fig. 5c). 6.2. Theory of ratcheting It is assumed here that a charging site, i.e. proteinaceous receptor tethers or lipid rafts, are distributed in a polarized mode. Several models for lipid rafts have been proposed (Anderson & Jacobson, 2002; Brown & London, 1998; Edidin, 2001; Simons & Toomre, 2000), enantioselective interactions play a role (Lalitha, Kumar, Stine, & Covey, 2001), and cellular and reconstituted rafts have been analyzed in size by viscous drag measurements and fluorescence microscopy, respectively (Dietrich et al., 2001; Pralle, Keller, Florin, Simons, & Horber et al., 2001). We consider the surface charge of lipid microdomains and entropic effects of ion pressure (Israelachvili & Wennerstrom, 1996) an important determinant of raft size, and introduce here the material property of lipid microdomains, the membrane dielectric, in our further considerations. Moreover, we assume that lipid rafts are self-contained entities that can be preferentially distributed despite continuous connections between luminal and abluminal bilayers. The process of transcellular transport could involve a Brownian ratchet (Reimann, 2002) and would possibly also toggle the receptor between two forms. It is furthermore assumed that ligand diffusion in three dimensions does not reduce transport in two dimensions to the negligible, and that the likelihood of pore traffic in two dimensions is higher than in three. A theory for a biological molecule is given in brief. The Brownian ratchet would concentrate the receptor on the luminal endothelial cell, raft side,

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delimit the starting point of walk upon release and transport the ligand. Ligand binding would trigger the partitioning of the receptor by a conformational change, by affecting the toggle, lower ligand-binding affinity and by altering the distribution of receptor between charging site and abluminal. The mechanism may require that the charging site is close to the abluminal side. In a proteinaceous ratchet scheme a refolding by diffusion in a receptor tether domain is proposed. This phenomenon is equivalent to the question whether two binding specificities can arise from a conformationally flexible protein domain, which is a likely scenario for proteins refolded in chaperones by mechanical energy. For a lipid binding ratchet, we envision rotation of amino acid side chains of the receptor (see also Duneau et al., 1998; Shen & Knutson, 2001; Szabo & Rayner, 1980) that are determined in energy level by correlation in response to the environment. The amino acid side chain atoms have steric interactions with each other and with the main chain. The embedding of main chains into the membrane bilayer has been discussed (White et al., 2001). Within the milieu of the bilayer with a low dielectric, the energy differences of dipolar rotational isomers of side chains are principally large compared to aqueous solvents, as shown for organic molecules as solutes with solvents (Abraham & Bretschneider, 1974), and in some respects bilayers behave as associated liquids (Herzfeld & Litovitz, 1959). The membrane dielectric constant, a determinant, is assumed to be rather homogenous within the horizontal plane of the membrane bilayer, alters vertically between headgroup area and hydrophobic core, and may generally alter inbetween different membrane microdomains of different lipid composition (Brockman, 1994). Measurements and calculations of the membrane dipole potential show that a diunsaturated glycerophospholipid has a higher dipole potential than a monounsaturated or saturated lipid (Gawrisch et al., 1992; Schamberger & Clarke, 2002). Simplifying to the hydrophobic core, we consider that kinked acyl chains have, e.g. a larger dipole moment than extended chains, and that the water contents of lipid compounds differ (Nagle & Tristram-Nagle, 2000). At the interface of two dielectrics, the receptor thus experiences an asymmetric potential, amino acid residues experience a structural change while partitioning which is released as a heat of binding upon entry into the raft domain,

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which concentrates the receptor. Moreover, quantized rotational energy levels in free rotation of some amino acid side chain residues in the different media differ, and diffusive broadening will drag the receptor into it. Receptor release from the lipid microdomain would be caused by a structural change of the protein due to ligand binding. In principle, this could change the tilt of an ␣-helix, or lead to altered hydrogen bonding of, e.g. a tyrosine at the membrane interface that would change energy levels of rotamers for energetically neutral partitioning. If a hydrophobic residue, initially buried within the protein, would be exposed to the hydrophobic membrane it would interact. As long as partitioning was not effective, the ligand–receptor complex would be in detailed equilibrium. Once partitioned, dissociation of ligand, that is, a lowered affinity constant in the non-raft domain, would follow from altered lipid headgroup surface charges and lipid structures, surrounding the protein, and collisions, which would alter a term akin to angular frequency of the metastable state (Kramers, 1940; Hänggi, Talkner, & Borkovec, 1990) and change the affinity constant. These lipid–protein interactions may be similar to previously described examples, but possibly not identical (Denisov, Wanaski, Luan, Glaser, & McLaughlin, 1998; Pigault, Follenius-Wund, & Chabbert, 1999; Martinez et al., 2002). This amounts to a scenario with three binding sites in the receptor, one for ligand, and one alternating structurally different binding site for lipids. The kinetics of partitioning of the receptor and off-rate of bound ligand determine the amount of “actively transported” ligand, and this process could increase ligand concentrations in the respective organ and relative to the blood stream (active transport), explained by this ratchet model of hop diffusion. Although this model is derived from a microscopical molecular consideration, it is similar to the purely mechanical molecular shuttle (Reimann, 2002). The source of Gaussian noise of fluctuations in our analogous model is due to a non-equilibrium state of the bilayer. The mechanical model of Marchesoni (Marchesoni, 1998), introducing boundary conditions in, e.g. the Kramers’ reaction rate (Kramers, 1940) (see also Reimann, 2002), leads to a current as explained by the Fokker–Planck equation. Due to the low vertical expansion of the cell monolayer of 100–300 nm, future imaging analysis of transcytosis will require ultrahigh resolution in the

z-direction. In vivo, transcellular transport includes blood flow as a further parameter. 6.3. Merocrine secretion The transcellular transport in endothelial cells of lymphoid origin utilizes a pathway involving lysosomes as an intermediate, in agreement with lysosomal vesicle budding and/or lysosomal-plasma membrane fusion (Reddy, Caler, & Andrews, 2001). The machinery for regulated secretion is present in endothelia and would be implicated in the latter event. This vesicular transport could mediate lipoprotein catabolism and reassembly, selective triacylglycerol, amino acid and cholesterol supply to the interstitium in general and/or lymph in reverse transport of modified lipoproteins to the liver. Alternatively, transport could be via amino acid transporters in the lysosomal and basal plasma membrane. Transport of some amino acids between the lysosomal lumen and cytoplasm has been found (Bernar et al., 1986; Pisoni, Acker, Lisowski, Lemons, & Thoene, 1990) and transporters have been localized to the apical and basal membrane of endothelia (Mann, Yudilevich, & Sobrevia, 2003). In lymphoid endothelia their existence and localization remains to be determined. For epithelial cells vectorial transport has been demonstrated (Nicklin, Irwin, Hassan, Mackay, & Dixon, 1995). Thus, uptake and degradation of LDL may contribute to amino acid uptake into organs from the blood in general and also to vectorial import.

Acknowledgements We thank Helena Bühler-Nurmi for technical assistance, Denise Paulin and Cora-Jean Edgell for cell lines, Takeshi Baba for the reagent and Richard Guggenheim for help with ESEM. We thank Kai Hencken for discussion and comments on the analogy to a mechanical shuttle. The Swiss National Science Foundation and Horten Foundation are acknowledged for financial support.

References Abraham, R. J., Bretschneider, E. (1974). Medium effects on rotational and conformational equilibria. In W J Orville-Thomas

E.S. Kuzmenko et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 519–534 (Ed.), Internal rotation in molecules (pp. 481–584). London: Wiley. Anderson, R. G. W. (1998). The caveolae membrane system. Annual Review in Biochemistry, 67, 199–225. Anderson, R. G., & Jacobson, K. (2002). A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science, 296, 1821–1825. Antohe, F., Poznansky, M. J., & Simionescu, M. (1999). Low density lipoprotein binding induces asymmetric redistribution of the low density lipoprotein receptors in endothelial cells. European Journal of Cell Biology, 78, 407–415. Baba, T., Rauch, C., Xue, M., Terada, N., Fujii, Y., Ueda, H., Takayama, I., Ohno, S., Farge, E., & Sato, S. B. (2001). Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly(ethylene glycol)-derivatized cholesterol in the plasma membrane. Traffic, 2, 501–512. Bernar, J., Tietze, F., Kohn, L. D., Bernardini, I., Harper, G. S., Grollman, E. F., & Gahl, W. A. (1986). Characteristics of a lysosomal membrane transport system for tyrosine and other neutral amino acids in rat thyroid cells. Journal of Biological Chemistry, 261, 17107–17112. Binder, H., & Gawrisch, K. (2001). Effect of unsaturated lipid chains on dimensions molecular order and hydration of membranes. Journal of Physical Chemistry, 105, 12378–12390. Borron, P., & Hay, J. B. (1994). Characterization of ovine lymphatic endothelial cells and their interactions with lymphocytes. Lymphology, 27, 6–13. Brockman, H. (1994). Dipole potential of lipid membranes. Chemical Physics and Lipids, 73, 57–79. Brown, F. L. H. (2003). Regulation of protein mobility via thermal membrane undulations. Biophysics Journal, 84, 842–853. Brown, D. A., & London, E. (1998). Functions of lipid rafts in biological membranes. Annual Review of Cell Development and Biology, 14, 111–136. Clough, G. (1991). Relationship between microvascular permeability and ultrastructure. Progress in Biophysics and Molecular Biology, 55, 47–69. Deane, R. et al., (2003). RAGE mediates amyloid-beta peptide transport across the blood–brain barrier and accumulation in brain. Natural Medicine, 9, 907–913. Dehouck, B., Fenart, L., Dehouck, M.-P., Pierce, A., Torpier, G., & Cecchelli, R. (1997). A new function for the LDL receptor: Transcytosis of LDL across the blood–brain barrier. Journal of Cell Biology, 138, 877–889. Denisov, G., Wanaski, S., Luan, P., Glaser, M., & McLaughlin, S. (1998). Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: An electrostatic model and experimental results. Biophysics Journal, 74, 731–744. Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson, K., & Gratton, E. (2001). Lipid rafts reconstituted in model membranes. Biophysics Journal, 80, 1417–1428. Dirks, W. G., MacLeod, R. A. F., & Drexler, H. G. (1999). ECV304 (endothelial) is really T24 (bladder carcinoma): Cell line cross-contamination at source. In Vitro Cell Development Biology of Animal, 35, 558–559.

531

Djoneidi, M., & Brodt, P. (1991). Isolation and characterization of rat lymphatic endothelial cells. Microcircular Endothelium Lymphatics, 7, 161–182. Mann, G. E., Yudilevich, D. L., & Sobrevia, L. (2003). Regulation of amino acid and glucose transporters in endothelial and smooth muscle cells. Physiological Review, 83, 183–252. Donald, A. M. (2003). The use of environmental scanning electron microscopy for imaging wet and insulating materials. Nature Materials, 2, 511–516. Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lauterbach, B., Menne, J., Lindschau, C., Mende, F., Luft, F. C., Schedl, A., Haller, H., & Kurzchalia, T. V. (2001). Loss of caveolae, vascular dysfunction and pulmonary defects in caveolin-1 gene-disrupted mice. Science, 293, 2449–2452. Duneau, J. P., Garnier, N., Cremel, G., Nullans, G., Hubert, P., Genest, D., Vincent, M., Gallay, J., & Genest, M. (1998). Time resolved fluorescence properties of phenylalanine in different environments. Comparison with molecular dynamics simulation. Biophysics and Chemistry, 73, 109–119. Dvorak, A. M., & Feng, D. (2001). The vesiculo-vacuolar organelle (VVO): A new endothelial cell permeability organelle. Journal of Histochemistry and Cytochemistry, 49, 419–431. Edgell, C. J. S., McDonald, C. C., & Graham, J. B. (1983). Permanent cell line expressing factor VIII related antigen established by hybridization. Proceedings of National Academy of Sciences of United States of America, 80, 3734–3737. Edidin, M. (2001). Shrinking patches and slippery rafts: Scales of domains in the plasma membrane. Trends in Cell Biology, 11, 492–496. Feng, D., Nagy, J. A., Hipp, J., Pyne, K., Dvorak, H. F., & Dvorak, A. M. (1997). Reinterpretation of endothelial cell gaps induced by vasoactive mediators in guinea-pig. Journal of Physiology, 504, 747–761. Fiedler, K., Lafont, F., Parton, R. G., & Simons, K. (1995). Annexin XIIIb: A novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. Journal of Cell Biology, 128, 1043–1053. Frokjaer-Jensen, J. (1980). Three-dimensional organization of plasmalemmal vesicles in endothelial cells. An analysis by serial sectioning of frog mesenteric capillaries. Journal of Ultrastructural Research, 73, 9–20. Galbiati, F., Volonte, D., Liu, J., Capozza, F., Frank, P. G., Zhu, L., Pestell, R. G., & Lisanti, M. P. (2001). Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism. Molecular and Biological Cell, 12, 2229–2244. Gawrisch, K., Ruston, D., Zimmerberg, J., Parsegian, V. A., Rand, R. P., & Fuller, N. (1992). Membrane dipole potentials, hydration forces, and the ordering of water at membrane surfaces. Biophysics Journal, 61, 1213–1223. Goldstein, J. L., Basu, S. K., & Brown, M. S. (1983). Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods in Enzymology, 98, 241–260. Goldstein, J. L., Basu, S. K., Brunschede, G. Y., & Brown, M. S. (1976). Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans. Cell, 7, 85–95.

532

E.S. Kuzmenko et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 519–534

Hajjar, D. P., & Haberland, M. E. (1997). Lipoprotein trafficking in vascular cells. Molecular Trojan horses and cellular saboteurs. Journal of Biological Chemistry, 272, 22975–22978. Hänggi, P., Talkner, P., & Borkovec, M. (1990). Reaction-rate theory: 50 years after Kramers. Review of Modern Physics, 62, 251–342. Hare, F., Amiell, J., & Lussan, C. (1979). Is an average viscosity tenable in lipid bilayers and membranes? A comparison of semi-empirical equivalent viscosities given by unbound probes: A nitroxide and a fluorophore. Biochimica Biophysica Acta, 555, 388–408. Havel, R. J., Eder, H. A., & Bragdon, J. H. (1955). The distribution and chemical composition of utracentrifugally separated lipoproteins in human serum. Journal of Clinical Investigation, 34, 1345–1353. Heimburg, T. (2000). Monte Carlo simulations of lipid bilayers and lipid protein interactions in the light of recent experiments. Current Opinion in Colloidal Interface Science, 5, 224–231. Herzfeld, K. F., Litovitz, T. A. (1959). Absorption and dispersion of ultrasonic waves. New York: Academic Press. Hianik, T., Haburcak, M., Lohner, K., Prenner, E., Paltauf, F., & Hermetter, A. (1998). Compressibility and density of lipid bilayers composed of polyunsaturated phospholipids and cholesterol. Colloid Surface A, 139, 189–197. Hough, G. P., Ross, L. A., Navab, M., & Fogelman, A. M. (1990). Transport of low density lipoproteins across endothelial monolayers. European Heart Journal, 11(Suppl. E), 62–71. Ishiwata, H., Sato, S. B., Vertut-Doi, A., Hamashima, Y., & Miyajima, K. (1997). Cholesterol derivative of poly(ethylene glycol) inhibits clathrin-independent, but not clathrin-dependent endocytosis. Biochimica Biophysica Acta, 1359, 123–135. Israelachvili, J., & Wennerstrom, H. (1996). Role of hydration and water structure in biological and colloidal interactions. Nature, 379, 219–225. Kamm, R. D. (2002). Cellular fluid mechanics. Annual Review of Fluid Mechanics, 34, 211–232. Kellen, M. R., & Bassingthwaighte, J. B. (2003). Transient transcapillary exchange of water driven by osmotic forces in the heart. American Journal of Physiology Heart Circulation and Physiology, 285, H1317–H1331. Kramers, H. A. (1940). Brownian motion in a field of force and the diffusion model of chemical reactions. Physica, 8, 284–304. Kucik, D. F., Elson, E. L., & Sheetz, M. P. (1999). Weak dependence of mobility of membrane protein aggregates on aggregate size supports a viscous model of retardation of diffusion. Biophysics Journal, 76, 314–322. Lalitha, S., Kumar, A. S., Stine, K. J., & Covey, D. F. (2001). Chirality in membranes: First evidence that enantioselective interactions between cholesterol and cell membrane lipids can be a determinant of membrane physical properties. Journal of Supramolecular Chemistry, 1, 53–61. Lang, T., Wacker, I., Wunderlich, I., Rohrbach, A., Giese, G., Soldati, T., & Almers, W. (2000). Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells. Biophysics Journal, 78, 2863–2877. Lipowsky, R. (1995). The morphology of lipid membranes. Current Opinion in Structural Biology, 5, 531–540.

Lipschultz, C. A., Yee, A., Mohan, S., Li, Y., & Smith-Gill, S. J. (2002). Temperature differentially affects encounter and docking thermodynamics of antibody-antigen association. Journal of Molecular Recognition, 15, 44–52. Lou, Y., Ge, M., & Freed, J. H. (2001). A multifrequency ESR study of the complex dynamics of membranes. Journal of Physical Chemistry B, 105, 11053–11056. Marchesoni, F. (1998). Conceptual design of a molecular shuttle. Physica Letters A, 237, 126–130. Marsh, D., & Horvath, L. I. (1998). Structure, dynamics and composition of the lipid–protein interface. Perspectives from spin-labelling. BBA, 1376, 267–296. Martinez, K. L., Gohon, Y., Corringer, P. J., Tribet, C., Merola, F., Changeux, J. P., & Popot, J. L. (2002). Allosteric transitions of Torpedo acetylcholine receptor in lipids. FEBS Letters, 528, 251–256. Michel, C. C., & Curry, F. E. (1999). Microvascular permeability. Physiological Review, 79, 703–761. Milici, A. J., Watrous, N. E., Stukenbrok, H., & Palade, G. E. (1987). Transcytosis of albumin in capillary endothelium. Journal of Cell Biology, 105, 2603–2612. Minshall, R. D., Tiruppathi, C., Vogel, S. M., Niles, W. D., Gilchrist, A., Hamm, H. E., & Malik, A. B. (2000). Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway. Journal of Cell Biology, 150, 1057–1069. Mostov, K., Su, T., & Ter Beest, M. (2003). Polarized epithelial membrane traffic: Conservation and plasticity. Nature and Cell Biology, 5, 287–293. Nagle, J. F., & Tristram-Nagle, S. (2000). Structure of lipid bilayers. BBA, 1469, 159–195. Nichols, B. J. (2003). GM1-containing lipid rafts are depleted within clathrin-coated pits. Current Biology, 13, 686–690. Nicklin, P. L., Irwin, W. J., Hassan, I. F., Mackay, M., & Dixon, H. B. (1995). The transport of acidic amino acids and their analogues across monolayers of human intestinal absorptive (Caco-2) cells in vitro. Biochimica Biophysica Acta, 1269, 176– 186. Niles, W. D., & Malik, A. B. (1999). Endocytosis and exocytosis events regulate vesicle traffic in endothelial cells. Journal of Membrane Biology, 167, 85–101. Nistor, A., & Simionescu, M. (1986). Uptake of low density lipoproteins by the hamster lung. Interactions with capillary endothelium. American Review of Respiration Disease, 134, 1266–1272. O’Connell, K. A., & Edidin, M. (1990). A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells. Journal of Immunology, 144, 521–525. Oh, P., McIntosh, D. P., & Schnitzer, J. E. (1998). Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium. Journal of Cell Biology, 141, 101–114. Palade, G. E. (1953). Fine structure of blood capillaries. Journal of Applied Physics, 24, 1424. Palade, G. E., & Bruns, R. R. (1968). Structural modulations of plasmalemmal vesicles. Journal of Cell Biology, 37, 633–653.

E.S. Kuzmenko et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 519–534 Parton, R. G. (1994). Ultrastructural localization of gangliosides GM1 is concentrated in caveolae. Journal of Histochemistry and Cytochemistry, 42, 155–166. Parton, R. G. (2003). Caveolae—from ultrastructure to molecular mechanisms. Nature and Review of Molecular Biology, 4, 162– 167. Pelkmans, L., Puntener, D., & Helenius, A. (2002). Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science, 296, 535–539. Pigault, C., Follenius-Wund, A., & Chabbert, M. (1999). Role of Trp-187 in the annexin V-membrane interaction: A molecular mechanics analysis. Biochemistry, Biophysics and Research Communication, 254, 484–489. Pisoni, R. L., Acker, T. L., Lisowski, K. M., Lemons, R. M., & Thoene, J. G. (1990). A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: Possible role in supporting lysosomal proteolysis. Journal of Cell Biology, 110, 327–335. Podgrabinska, S., Braun, P., Velasco, P., Kloos, B., Pepper, M. S., Jackson, D. G., & Skobe, M. (2002). Molecular characterization of lymphatic endothelial cells. Proceedings of National Academy of Sciences of United States of America, 99, 16069–16074. Pralle, A., Keller, P., Florin, E.-L., Simons, K., & Horber, J. K. H. (2001). Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. Journal of Cell Biology, 148, 997–2007. Predescu, D., Predescu, S., McQuistan, T., & Palade, G. E. (1998). Transcytosis of alpha(1)-acidic glycoprotein in the continuous microvascular endothelium. Proceedings of National Academy of Sciences of United States of America, 95, 6175–6180. Razani, B., Engelman, J. A., Wang, X. B., Schubert, W., Zhang, X. L., Marks, C. B., Macaluso, F., Russell, R. G., Li, M., Pestell, R. G., Di Vizio, D., Hou, H., Kneitz, B., Lagaud, G., Christ, G. J., Edelman, W., & Lisanti, M. P. (2001). Caveolin-1 null mice are viable but show evidence of hyper-proliferative and vascular abnormalities. Journal of Biological Chemistry, 276, 38121–38138. Reddy, A., Caler, E. V., & Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell, 106, 157–169. Reimann, P. (2002). Brownian motors: Noisy transport far from equilibrium. Physical Reports, 361, 57–265. Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., & Deisenhofer, J. (2002). Structure of the LDL receptor extracellular domain at endosomal pH. Science, 298, 2353–2358. Saffman, P. G., & Delbruck, M. (1975). Brownian motion in biological membranes. Proceedings of National Academy of Sciences of United States of America, 72, 3111–3113. Sandvig, K., & van Deurs, B. (2002). Membrane traffic exploited by protein toxins. Annual Review of Cell Development and Biology, 18, 1–24. Saxton, M. J., & Jacobson, K. (1997). Single-particle tracking: Application to membrane dynamics. Annual Review of Biophysics and Biomolecular Structures, 26, 373–399.

533

Schamberger, J., & Clarke, R. J. (2002). Hydrophobic ion hydration and the magnitude of the dipole potential. Biophysics Journal, 82, 3081–3088. Schrader, W., Ebel, H., Grabitz, P., Hanke, E., Heimburg, T., Hoeckel, M., Kahle, M., Wente, F., & Kaatze, U. (2002). Compressibility of lipid mixtures studied by calorimetry and ultrasonic velocity measurements. Journal of Physical Chemistry B, 106, 6581–6586. Schmid, S. L., & Smythe, E. (1991). Stage-specific assays for coated pit formation and coated vesicle budding in vitro. Journal of Cell Biology, 114, 869–880. Schnitzer, J. E., Oh, P., & McIntosh, D. P. (1996). Role of GTP hydrolysis in fission of caveolae directly from plasma membranes. Science, 274, 239–242. Schnitzer, J. E., Oh, P., Pinney, E., & Allard, J. (1994). Filipin-sensitive caveolae-mediated transport in endothelium: Reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. Journal of Cell Biology, 127, 1217–1232. Seifert, U. (1997). Configurations of fluid membranes and vesicles. Advanced Physics, 46, 13–137. Shen, X., & Knutson, J. R. (2001). Excited states subpicosecond fluorescence spectra of tryptophan in water. Journal of Physical Chemistry B, 105, 6260–6265. Simionescu, M., & Simionescu, N. (1991). Endothelial transport of macromolecules: Transcytosis and endocytosis. Cell Biology and Review, 25, 1–80. Simionescu, N., Simionescu, M., & Palade, G. E. (1975). Permeability of muscle capillaries to small heme-peptides. Journal of Cell Biology, 64, 586–607. Simons, K., & Toomre, D. (2000). Lipid rafts and signal transduction. Nature and Review of Molecular Cell Biology, 1, 31–39. Simons, K., & van Meer, G. (1988). Lipid sorting in epithelial cells. Biochemistry, 27, 6197–6202. Simson, R., Yang, B., Moore, S. E., Doherty, P., Walsh, F. S., & Jacobson, K. A. (1998). Structural mosaicism on the submicron scale in the plasma membrane. Biophysics Journal, 74, 297– 308. Stolz, D. B., & Jacobson, B. S. (1992). Examination of transcellular membrane protein polarity of bovine aortic endothelial cells in vitro using the cationic colloidal silica microbead membrane-isolation procedure. Journal of Cell Sciences, 103, 39–51. Szabo, A. G., & Rayner, D. M. (1980). Fluorescence decay of tryptophan conformers in aqueous solution. Journal of American Chemical Society, 102, 554–563. Takahashi, K., Sawasaki, Y., Hata, J., Mukai, K., & Goto, T. (1990). Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell Developmental Biology, 26, 265–274. Tomishige, M., & Kusumi, A. (1999). Compartmentalization of the erythrocyte membrane by the membrane skeleton: Intercompartmental hop diffusion of band 3. Molecular and Biological Cell, 10, 2475–2479. Tsuji, A., & Ohnishi, S. (1986). Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network:

534

E.S. Kuzmenko et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 519–534

Dependence on spectrin association state. Biochemistry, 25, 6133–6139. Uehara, K., & Miyoshi, M. (1999). Tight junction of sinus endothelial cells of the rat spleen. Tissue Cell, 31, 555–560. Van Deurs, B., Roepstorff, K., Hommelgaard, A. M., & Sandvig, K. (2003). Caveolae: Anchored, multifunctional platforms in the lipid ocean. TiCB, 13, 92–100. Vasile, E., Simionescu, M., & Simionescu, N. (1983). Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ. Journal of Cell Biology, 96, 1677–1689. Wang, N., Naruse, K., Stamenovic, D., Fredberg, J. J., Mijailovich, S. M., Tolic-Norrelykke, I. M., Polte, T., Mannix, R., &

Ingber, D. E. (2001). Mechanical behavior in living cells consistent with the tensegrity model. Proceedings of National Academy of Sciences of United States of America, 98, 7765– 7770. White, S. H., Ladokhin, A. S., Jayasinghe, S., & Hristova, K. (2001). How membranes shape protein structure. Journal of Biological Chemistry, 276, 32395–32398. Wilks, S. S. (1948). Elementary statistical analysis. Princeton, NJ: Princeton University Press. Willnow, T. E., Nykjaer, A., & Herz, J. (1999). Lipoprotein receptors: New roles for ancient proteins. Nature and Cell Biology, 1, E157–62.