Extra Membrane Research Articles

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of gram-negative bacteria are also present in other mollicutes and gram-positive bacteria.

Requirement of TCF7L2 for TGF-β-dependent Transcriptional Activation of the TMEPAI Gene

The TGF-β and Wnt pathways are involved in cell fate and tumorigenicity. A recent report indicated that a TGF-β target gene, TMEPAI (transmembrane prostate androgen-induced RNA), is possibly also a downstream target of Wnt signaling. Although TMEPAI was believed to be involved in tumorigenicity because of its blockage of TGF-β signaling, how TGF-β and Wnt signals affect the activation of the TMEPAI gene is not well understood. Herein, we show that the TMEPAI promoter is regulated synergistically by TGF-β/Smad and Wnt/β-catenin/T cell factor (TCF) 7L2. The critical cis-element for dual signals, termed TGF-β-responsive TCF7L2-binding element (TTE), is located in intron 1 of the TMEPAI gene. TCF7L2, but not Smad proteins, bound to TTE, whereas the disruption of TTE by mutagenesis remarkably counteracted both TGF-β and TCF7L2 responses. The introduction of mutations in critical Smad-binding elements blocked the activation of the TMEPAI promoter by TCF7L2. Furthermore, our DNA-protein interaction experiments revealed the indirect binding of TCF7L2 to Smad-binding elements via Smad3 upon TGF-β stimulation as well as its TGF-β-dependent association with TTE. We demonstrate that the Wnt/β-catenin/TCF7L2 pathway is preferentially able to alter the transcriptional regulation of the TGF-β-target gene, TMEPAI.

A comparison study on histopathology and ultrastructure of lesions from patients with bullous ichthyosiform erythrodermia before and after treatment with acitretin.

Objective To investigate the effect of acitretin on the histopathology and ultrastructure of lesions from patients with bullous ichthyosiform erythrodermia (BIE), and to explore mechanisms underlying the modulation of keratinization process by acitretin. Methods Lesional tissue was obtained from the back of 4 patients with BIE before and after the treatment with acitretin. Light microscopy and transmission electron microscopy were performed to observe histopathological and ultrastructural changes in these lesions. Results After treatment, the improvement in clinical manifestations was more than 75% in all the 4 patients, and reached 90% in 1 of the 4 patients. As histopathology and ultrastructural study showed, there was an obvious improvement in hyperkeratosis and continuity of extra cellular lamellar membrane, and a decrease in keratin deposition in prickle and granular layer, but no remarkable changes were observed for the proliferation of prickle cells or acantholysis. Conclusions Acitretin shows a favorable efficacy in clinical treatment of BIE,with histopathological and ultrastructural improvement mainly located in the stratum corneum. The modulation of keratinization process in keratinocytes by acitretin appears more apparent in granular and corneum layers. Key words: Ichthyosis; Acitretin; Microscopy,electron,transmission

Removal of drugs of abuse from municipal wastewater using reverse osmosis membranes

Drugs of abuse are important emerging contaminants due to their presence in water bodies following incomplete removal in wastewater treatment. This poses a threat to drinking water sources and has led to concerns about possible health effects, both to wildlife, ecosystems and humans. In recent years, there has been growing interest in the incorporation of reverse osmosis (RO) and nanofiltration (NF) membrane technologies into existing municipal and industrial wastewater treatment facilities as a quaternary treatment option. By improving effectiveness of wastewater treatment, the exposure of firstly, nature and secondly, humans to these compounds can be minimized. It has been suggested that these membrane techniques are suitable for cost-effective desalination and the removal of a wide range of low-molecular-weight (LMW) trace organic constituents, including drugs of abuse. This paper presents the detailed results of a feasibility assessment study on the removal of selected drugs and metabolites from secondary treated waste-water. The study was carried out at a small scale RO pilot plant. Three different FILMTECTM brackish water membranes were used; low energy (LE) membrane, high rejection membrane (BW30) and extra fouling resistant membrane (XFR). The results show that RO membranes can reduce the amount of target compounds in the effluent water. The obtained mean rejection values for the three membranes and six monitoring campaigns were as follows: 74–83% (caffeine), 49–63% (nicotine), 94–96% (cotinine), 98–99% (codeine), 98% (norcodeine), 81% (METH), 57–64% (MDA), 93–96% (MDMA) and 47–57% (MDEA). No major differences were observed between the three different membranes. This indicates that the low energy RO membrane provides the same removal efficiency as the other two membranes, thus demonstrating the potential for a less energy intensive RO plant operation.

Recycling Endosome Membrane Incorporation into the Leading Edge Regulates Lamellipodia Formation and Macrophage Migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R-SNARE VAMP3 on the recycling endosome partnering with the surface Q-SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3-mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3-mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.

Formation of the Fertilization Pore during Oogenesis of the Fern Ceratopteris thalictroides

The development of the fertilization pore during oogenesis of the fern Ceratopteris thalictroides was followed using transmission electron microscopy. The newly formed egg is appressed closely to the adjacent cells. There are well-developed plasmodesmata between the egg and the ventral canal cell, but none between the egg and the jacket cells of the archegonium. During maturation, a separation cavity is formed around the egg. However, a pore region persistently connects the egg and the ventral canal cell. The extra egg membrane is formed by deposition of sheets of endoplasmic reticulum (ER), but no ER is deposited on the inner surface of the pore region. Thus, a fertilization pore, covered by a layer of plasmalemma, is formed. The ventral canal cell undoubtedly participates the formation of the fertilization pore, probably by absorbing the sheets of ER beneath the pore region. The functional significance of the ventral canal cell in formation of the fertilization pore is discussed. The features of the mature egg include that abundant concentric membranes and osmiophilic vesicles occur in the cytoplasm of the mature egg. The initial, round nucleus of the egg eventually becomes cup-shaped. This investigation gives some new insights about the cells participating oogenesis in ferns.

Antibacterial Properties of a Kind of Schiff Base and Its Neodymium(III) and Zn(II) Complex (ZnNdL) on Escherichia coli

AbstractThe microcalorimetric and electronic microscopy methods were used to study the antibacterial activity of a Schiff base and its complex ZnNdL on Escherichia coli. The metabolic power‐time curves of the bacteria treated with the compounds were obtained, and the thermokinetic parameters were analyzed. The results show that the compounds (H2L, ZnNdL) have good activity on aerobic multiplying metabolism of E. coli, with the values of IC50 57.0 and 54.4 mg·L−1, respectively. In order to further investigate their mechanism on E. coli, transmission electronic microscopy and scanning electronic micrography were used to study the cell membrane change induced by the dibasic quadridentate Schiff base and its neodymium(III) complex. At a low concentration of the compound, the flagellum was inhibited and the cell did not show distinct changes. However, the flagella around the cell membrane were exfoliated, the morphology of E. coli was changed from a rod shape into a spherical shape or a short rod, and the flagella disappeared completely at a high concentration. The drug permeability into cell membrane was investigated by fluorescence quenching of probe dis‐C3‐(5), which suggested that H2L and ZnNdL could influence on the intra and extra cell membrane.

Lats kinase is involved in the intestinal apical membrane integrity in the nematodeCaenorhabditis elegans

The roles of Lats kinases in the regulation of cell proliferation and apoptosis have been well established. Here we report new roles for Lats kinase in the integrity of the apical membrane structure. WTS-1, the C. elegans Lats homolog, localized primarily to the subapical region in the intestine. A loss-of-function mutation in wts-1 resulted in an early larval arrest and defects in the structure of the intestinal lumen. An electron microscopy study of terminally arrested wts-1 mutant animals revealed numerous microvilli-containing lumen-like structures within the intestinal cells. The wts-1 phenotype was not caused by cell proliferation or apoptosis defects. Instead, we found that the wts-1 mutant animals exhibited gradual mislocalization of apical actin and apical junction proteins, suggesting that wts-1 normally suppresses the formation of extra apical membrane structures. Heat-shock-driven pulse-chase expression experiments showed that WTS-1 regulates the localization of newly synthesized apical actins. RNAi of the exocyst complex genes suppressed the mislocalization phenotype of wts-1 mutation. Collectively, the data presented here suggest that Lats kinase plays important roles in the integrity of the apical membrane structure of intestinal cells.

On the origin of chloroplasts, import mechanisms of chloroplast-targeted proteins, and loss of photosynthetic ability — review

Primary plastids of green algae (including land plants), red algae and glaucophytes are bounded by two membranes and are thought to be derived from a single primary endosymbiosis of a cyanobacterium in a eukaryotic host. Complex plastids of euglenids and chlorarachneans bounded by three and four membranes, respectively, most likely arose via two separate secondary endosymbioses of a green alga in a eukaryotic host. Secondary plastids of cryptophyta, haptophyta, heterokontophyta and apicomplexan parasites bounded by four membranes, and plastids of dinoflagellates bounded by three membranes could have arisen via a single secondary endosymbiosis of a red alga in a eukaryotic host (chromalveolate hypothesis). However, the scenario of separate tertiary origins (symbioses of an alga possessing secondary plastids in a eukaryotic host) of some (or even most) chromalveolate plastids can be also consistent with the current data. The protein import into complex plastids differs from the import into primary plastids, as complex plastids contain one or two extra membrane(s). In organisms with primary plastids, plastid-targeted proteins contain N-terminal transit peptide which ferries proteins through the protein import machineries (multiprotein complexes) of the two (originally cyanobacterial) membranes. In organisms with complex plastids, the secretory signal sequence directing proteins to endomembrane system and afterwards through extra outermost membrane(s) is generally present upstream of the classical transit peptide. Several free-living as well as parasitic eukaryotes possess non-photosynthetic plastids. These plastids have generally retained the plastid genome, functional plastid transcriptional and translational apparatus, and various metabolic pathways, suggesting that though these plastids lost their photosynthetic ability, they are essential for the mentioned organisms. Nevertheless, some eukaryotes could have lost chloroplast compartment completely.

Wortmannin induces homotypic fusion of plant prevacuolar compartments*

Wortmannin, a specific inhibitor of phosphatidyl-inositol 3-kinase, is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. It has recently been demonstrated that wortmannin at 16.5 μM or 33 μM caused the prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs) by their enrichment in vacuolar sorting receptor (VSRs) proteins and the BP-80 reporter, to form small vacuoles rapidly. However, the source(s) of the membrane needed for the rapid enlargement of PVCs/MVBs has been unclear. Using both confocal immunofluorescence and immunogold EM with high pressure freeze substitution of plant samples, it has been demonstrated here that wortmannin induces homotypic fusions of PVCs/MVBs thus providing an explanation for the demand for extra membrane. In addition, possible wortmannin-induced fusions between the trans-Golgi network (TGN) and PVC, as well as between the small internal vesicles and PVC membrane, were also observed and they may also contribute to the membranes needed for PVC enlargement. In contrast to mammalian cells and yeast, wortmannin-induced fusion of PVCs appears to be unique to plants.

Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System

Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with alpha2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma.

Discovery of Porcine microRNAs and Profiling from Skeletal Muscle Tissues during Development

MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.

Membrane technology for sustainable treated wastewater reuse: agricultural, environmental and hydrological considerations

Field experiments were conducted in agricultural fields in which secondary wastewater of the City of Arad (Israel) is reused for irrigation. For sustainable agricultural production and safe groundwater recharge the secondary effluent is further polished by a combined two-stage membrane pilot system. The pilot membrane system consists of two main in row stages: Ultrafiltration (UF) and Reverse Osmosis (RO). The UF stage is efficient in the removal of the pathogens and suspended organic matter while the successive RO stage provides safe removal of the dissolved solids (salinity). Effluents of various qualities were applied for agricultural irrigation along with continuous monitoring of the membrane system performance. Best agricultural yields were obtained when applying effluent having minimal content of dissolved solids (after the RO stage) as compared with secondary effluent without any further treatment and extended storage. In regions with shallow groundwater reduced soil salinity in the upper productive layers, maintained by extra membrane treatment, will guarantee minimal dissolved solids migration to the aquifers and minimize salinisation processes.

A novel configuration of microbial fuel cell stack bridged internally through an extra cation exchange membrane

This paper reports a novel configuration of stacked microbial fuel cells (MFCs) bridged internally through an extra cation exchange membrane (CEM). The MFC stack (MFC(stack)), assembled from two single MFCs (MFC(single)), resulted in double voltage output and half optimal external resistance. COD removal rate was increased from 32.4% to 54.5%. The performance improvement could be attributed to the smaller internal resistance and enhanced cations transfer. A result from a half cell study further confirmed the important role of the extra CEM. This study also demonstrated MFCs where the anode and cathode were sandwiched between two CEMs possessed significantly high power outputs.

The Cytotoxic Lipid Peroxidation Product 4-Hydroxy-2-nonenal Covalently Modifies a Selective Range of Proteins Linked to Respiratory Function in Plant Mitochondria

Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments.

The structural basis of novel endosome anchoring activity of KIF16B kinesin

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions.

Laser capture microdissection and cDNA microarrays used to generate gene expression profiles of the rapidly expanding fibre initial cells on the surface of cotton ovules

Cotton (Gossypium hirsutum L.) fibre initial cells undergo a rapid cellular re-programming around anthesis to form the long cellulose fibres prized for textile manufacture. On the day of anthesis the cotton fibre initial cells balloon out from the ovule surface and so are clearly distinguished from adjacent epidermal pavement cells. To enhance our understanding of the molecular processes that determine which cells become fibres and why adjacent epidermal cells remain in a different developmental state we studied the expression profiles of the two respective cell types. Using laser-capture microdissection, coupled with an in vitro RNA amplification system, we used cDNA microarray slides to profile the gene expression in expanding fibre initials compared to the non-expanding epidermal cells at an early stage just after the fibre initials are discernable. Except for a few regulatory genes, the genes that are up-regulated in the cotton fibre initials relative to epidermal cells predominantly encode proteins involved in generating the components for the extra cell membrane and primary cell wall needed for the rapid cell expansion of the initials. This includes synthesis of enzymes and cell wall proteins, carbohydrates, and lipids. An analysis of single channel fluorescence levels confirmed that these classes of genes were also the most highly expressed genes in fibre initials. Genes involved in DNA metabolism were also well represented in the expanding fibre cell, consistent with the limited endoreduplication we previously reported to occur in fibre initial cells.

Pinched off membrane

Cells slough off extra membrane by pinching it off into the extracellular space, say Dubreuil et al. on page 483. The discarded membrane can be released by either cilia or midbodies, with potentially distinct outcomes. Figure 1 Prominin-1 (red) and a midbody marker (green) are found on midbodies (arrowheads) and in lumenal particles (inset). Membrane particles can be seen floating in the extracellular space of the developing neural tube. These particles are distinct from signaling exosomes, leaving their origin in question. The authors now show that one origin of these particles is the midbody—the membrane structure that transiently connects two daughter cells and contains the contractile ring. The group identified the midbody as the source by examining EMs of neural tissue serial sections. They showed that particles emerge from midbodies of dividing neuroepithelial cells. The midbody particles are released at a developmental stage when these cells switch from symmetric, proliferative divisions to the asymmetric, differentiation-inducing divisions that produce neurons. Differentiation might be a consequence of particle release, as the discarded membrane is enriched in a stem cell marker called Prominin-1. If this protein confers a stem cell phenotype, its irreversible expulsion might drive differentiation. The authors are now testing whether cancer cells have lost the ability to discard the Prominin-1 particles, thus preserving the stem cell-like state. At the onset of neurogenesis, neuroepithelial cells released Prominin-1 particles that are smaller than midbodies and appear to originate instead from cilia. Their release might help to shrink the membrane of the cilium, which disappears during mitosis. The authors speculate that free-floating Prominin-1 bodies might also relay signaling information. Perhaps, for instance, the particles notify neighbors that a division has taken place as part of a size control mechanism. The components that make up this new membrane disposal pathway are still unknown, but they should be widely expressed; preliminary results indicate that epidermal and mesenchymal tissues also release midbody particles.

Membrane Reserves and Hypotonic Cell Swelling

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.