A possible role of cholesterol in membrane adhesion

Calcium phosphate induced membrane aggregation was studied for erythrocyte vesicles and lipid membrane vesicles. The later lipid membrane components were similar in composition to those of erythrocyte membranes. The presence of an appropriate amount of cholesterol is an important factor in the production of the calcium phosphate dependent membrane aggregation.     

Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM

The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by

1. 1. [³⁵S]methionine internal radiolabeling;

2. 2. [¹²⁵I]iodine labeling by a lactoperoxidase-mediated method;

3. 3. [³H]fucose internal labeling;

4. 4. binding to a lentil lectin adsorbant column;

5. 5. susceptibility to digestion with limited amounts of papain.

Of the three methods of radiolabeling membrane proteins, [³⁵S]methionine best displayed all proteins although some individual proteins were heavily iodinated or fucosylated. Thirty proteins were externally exposed as defined by susceptibility to lactoperoxidase-mediated radio-iodination and to digestion with minute amounts of papain. Thirtyfive proteins were bound to a lentil lectin absorbant column. p44 (HLA-A and -B antigens) were iodinated, fucosylated, susceptible to papain digestion and bound to the lectin column. β₂-Microglobulin was iodinated and bound to the lectin column. The identifications and functions of other membrane proteins were not known. In general, proteins of high molecular weight (100 000 to 250 000 D) were more heavily radio-iodinated and fucosylated than were proteins of lower molecular weights. p95 was the most heavily fucosylated protein, p110, which had been identified only on T lymphoblasts, was fucosylated and was iodinated. p65, which was found only on the T lymphoblast line CCRF-CEM and could represent a lymphocyte subpopulation-specific molecule, was iodinated and fucosylated. p15 and p18 were equally and densely labeled with [³⁵S]methionine but only p18 was fucosylated and it was heavily radio-iodinated. These experiments help to define the external membrane proteins of a T lymphoblast cell line in part for the selection of proteins for isolation in order to raise antisera for immunodiagnostic and functional studies.     

ERM proteins in cell adhesion and membrane dynamics.

Ezrin, radixin and moesin, collectively known as the ERM proteins, are a group of closely related membrane-cytoskeleton linkers that regulate cell adhesion and cortical morphogenesis. ERM proteins can self-associate through intra- and inter-molecular interactions, and these interactions mask several binding sites on the proteins. ERM activation involves unfolding of the molecule, and allows the protein to bind to plasma membrane components either directly, or indirectly through linker proteins. The discovery that the tumour-suppressor NF2, also known as merlin/schwannomin, is related to ERM proteins has added a new impetus to investigations of their roles. This review discusses current understanding of the structure and function of members of the ERM family of proteins.     

Reactions of peroxynitrite with globin proteins and their possible physiological role

It is now widely accepted that, besides their well-established function in O(2) transport, hemoglobin and myoglobin also undergo several redox reactions aimed to scavenge toxic free radicals and reactive oxygen and nitrogen species. At least some of these reactions are believed to play an important physiological role in the defense against oxidative stress. This aspect is exemplified by the recently discovered neuroglobin, a globin expressed in the brain. Rather than being considerably involved in reversible O(2) binding, neuroglobin is likely to undergo redox reactions to protect neurons against oxidative and potentially pathogenic pathways, as those operating after episodes of tissue hypoxia or ischemia. A major part of the cellular damage occurring under such conditions has been ascribed to formation of peroxynitrite, that originates from the reaction between two biologically important free radicals, nitric oxide (NO ) and superoxide. Here we review the current knowledge of the reactions of different forms of hemoglobin, myoglobin, and neuroglobin with peroxynitrite and discuss their physiological role on the basis of measured rate constants and on the probability of occurrence of these reactions in vivo.     

A role for fibronectin-leucine-rich transmembrane cell-surface proteins in homotypic cell adhesion

The fibronectin-leucine-rich transmembrane (FLRT) family of leucine-rich repeat (LRR) proteins is implicated in fibroblast growth factor (FGF) signalling, early embryonic development and neurite outgrowth. Here, we have analysed whether FLRTs may also function in cell adhesion. We find that FLRT proteins can physically interact and that FLRT-transfected cultured cells sort out from non-transfected cells, suggesting a change in adhesive properties. A similar sorting effect is also observed in Xenopus embryos and tissue aggregates. FLRT-mediated cell sorting is calcium dependent and substrate independent. Deletion analysis indicates that cell sorting requires the LRR domains, which are dispensable for FLRT-mediated FGF signalling. Conversely, sorting is independent of the cytoplasmic domain, which is essential for FLRT-induced signalling. Therefore, FLRT-mediated FGF signal transduction and homotypic cell sorting can be molecularly uncoupled. The results indicate that FLRT proteins have a dual role, promoting FGF signalling and modulating homotypic cell adhesion.     

Stimulation of macrophage by polyanions and its conjugated proteins and effect on cell membrane

Lipophilic anionic copolymer (styrene-maleic acid; SMA) conjugates of albumin and antitumor protein neocarzinostatin (NCS) (smancs) were found to stimulate the release of H2O2 and O-2 from the peritoneal macrophages obtained from mice which had been pretreated with the heat-killed preparation of Streptococcus pyogenes (OK-432) in vivo. Some alkyl esters of SMA exhibited effects similar to protein-polymer conjugates. Among them, butyl-SMA was the most effective followed by ethyl-SMA, whereas hydrolyzed SMA showed no effect. This activity was dose-dependent but exhibited a bell-shape profile. These results suggest that the aliphatic ester residue in SMA as well as the main chain of the copolymer may be important for the activation of macrophages. A strong antitumor effect of smancs reported elsewhere may be attributed partly to the activation of macrophages in addition to the direct damage to the cellular DNA by the NCS component. A preliminary investigation of the subcellular mechanism of macrophage activation was carried out in view of membrane fluidity by the fluorescence polarization method. The results showed that the apparent decrease in the cell membrane fluidity and the degree of macrophage activation paralleled the same dose range and at similar time courses. This indicated the interaction of SMA component and macrophage cell membrane.     

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.     

A possible role of hydroxyproline-containing proteins in the cessation of cell elongation

Hydroxyproline-containing proteins increase markedly in the cell walls of the Alaska pea epicotyl during the transition of rapidly elongating tissue into non-elongating, mature tissue. The increase in these proteins may be a factor in the cessation of cell elongation.     

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.     

The role of human red blood cell membrane skeletal proteins in repetitive membrane strain and rupture

The influence of the membrane skeleton on cell membrane deformability, elasticity, and rupture after repetitive cycles of membrane strain, release and rupture was investigated. Human red blood cells were electrofused to doublets, which showed the post-fusion oscillation-movement. Geometrical developments of heat-treated cells were measured and compared to control cells. Alterations of cluster length and fusion zone diameter during repetitive colloidosmotic swelling period grow with heat treatment, and the number of precedent swell phases has minor influence on these values. Irrespective of the treatment, the geometrical doublet configuration at which a membrane rupture is initiated has an almost constant roundness index of 0.89. Increasing heat treatment temperature was shown to affect both deformability and elasticity of the membrane, such that doublets start each swell phase of the oscillation cycle from decreased roundness values. Evidence is given that there is a difference in the mechanical properties between the membrane at the fusion zone and the membrane of native red blood cells.     

Arf GTPase-activating proteins and their potential role in cell migration and invasion

Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.Key words: Arf GAP, Arf, effector, ADP-ribosylation factor, GTPase-activating protein, focal adhesions, podosomes, invadopodia, cell migrationCell migration involves adhesive structures in which the cell membrane is integrated with the actin cytoskeleton.¹ Cells acquire a spatial asymmetry to enable them to turn intracellular generated forces into a net cell body translocation. With the asymmetry, there is a clear distinction between the cell front and rear. Active membrane processes, including lamellipodia and filopodia, take place primarily around the cell front. Extension of both filopodia and lamellipodia is coupled with local actin polymerization, which generates protrusive force. In some cells, focal complexes form at the leading edge of lamellipodia and filopodia. Focal complexes are specialized surfaces of the plasma membrane that mediate attachment to the substratum, providing traction and allowing the cell edge to protrude. Focal complexes mature with cell migration to form another specialized surface in the plasma membrane, focal adhesions (FAs). FAs localize to the termini of stress fiber bundles and serve in longer-term anchorage at the rear of the cell.² A contractile force is generated at the rear of the cell by the myosin motors to move the cell forward and cell-substratum (extracellular matrix) attachments are released to retract the cell rear. In some cells, podosomes are adhesive structures that mediate cell migration and sometimes invasion.The structures involved in cell migration that are affected by Arf GAPs are FAs, podosomes and invadopodia. FAs contain multiple proteins, including integrins, which are transmembrane proteins.³ The extracellular part of integrins binds to the extracellular matrix. The cytoplasmic domains of integrins associate with multiple signaling proteins as well as proteins that are part of the actin cytoskeleton, thereby coordinating signaling events involved in cell migration and linking the extracellular matrix to the cytoskeleton. Cytoplasmic proteins critical to the function of FAs and that are often used as markers of FAs include vinculin, paxillin and focal adhesion kinase. At least five distinct Arf GAPs have been found to associate with FAs, including GIT1, GIT2, ASAP1, ASAP3 and ARAP2.⁴Podosomes and invadopodia are related structures induced by action of Src (reviewed in ref. ⁵). They contain some proteins in common with FAs, but do have some differences that likely reflect different function and/or regulation. For example, podosomes contain ASAP1 but not ASAP3.⁶ Podosomes and invadopodia have not been examined for the presence of other Arf GAPs. Like FAs, podosomes and invadopodia mediate adhesion to extracellular surfaces. In addition, they are points of degradation of the extracellular matrix and may transfer tension along the extracellular matrix to enable the cell to move. Consistent with the function in motility, podosomes and invadopodia are dynamic structures, turning over in minutes. Podosomes are found in normal physiology of cells including smooth muscle cells, osteoclasts and macrophages and in Src-transformed fibroblasts. Invadopodia are observed in transformed cells, such as cells derived from breast cancers.Two families of GTP-binding proteins within the Ras superfamily, Rho and Arf, are involved in both actin and membrane remodeling. RhoA regulates stress fibers (bundles of actin filaments that traverse the cell and are linked to the extracellular matrix through FAs) and the assembly of FAs.⁷ Rac1 regulates membrane ruffling and lamellipodia formation.⁸ Cdc42 regulates filopodia formation.⁹ Activation of Cdc42 has been shown to lead to the sequential activation of Rac1 and then RhoA in growth factor stimulated fibroblasts.Arf proteins regulate membrane traffic and the actin cytoskeleton.¹⁰ There are six mammalian Arf proteins, divided into three classes based on their amino-acid sequence. Arf12 and 3 are class I, Arf4 and Arf5 are class II and Arf6 is the single member of the class III group. Arf1 and Arf6 have been the most extensively studied. Most work has focused on Arf1 function in the Golgi apparatus and endocytic compartments although Arf1 has been found to affect paxillin recruitment to FAs and trafficking of epidermal growth factor receptor from the plasma membrane. Arf6 affects the endocytic pathway and the peripheral actin cytoskeleton.The function of Rho and Arf family proteins depends on a cycle of binding and hydrolyzing GTP. However, Rho and Arf family proteins have slow intrinsic nucleotide exchange. Rho family proteins have slow intrinsic GTPase activity and Arf family proteins have no detectable intrinsic GTPase activity. The cycle of GTP binding and hydrolysis is driven by accessory proteins called guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho family proteins are also regulated by guanine nucleotide dissociation inhibitors, which prevent spontaneous activation in the cytoplasm.Arf GAPs are enzymes that catalyze the hydrolysis of GTP bound to Arf proteins, thereby converting Arf•GTP to Arf•GDP. Thirty-one genes in human encode proteins with Arf GAP domains (Fig. 1). The Arf GAP family is divided into ten subgroups based on domain structure and phylogenetic analysis.¹¹ Six subgroups contain the Arf GAP domain at the N-terminus of the protein. Four groups contain a tandem of a PH, Arf GAP and Ankyrin repeat domains. The Arf GAP nomenclature is mostly based on the protein domain structure. For instance, the ASAP first identified, ASAP1, contains Arf GAP, SH3, Ank repeat and PH domains; ARAPs contain Arf GAP, Rho GAP, Ank repeat and PH domains; ACAPs contain Arf GAP, coiled-coil (later identified as BAR domain), Ank repeat and PH domains; and AGAPs contain Arf GAP, GTP-binding protein-like, Ank repeat and PH domains.Open in a separate windowFigure 1Domain structure of the Arf GAP family. The schematic representation of the ten groups of proteins containing the Arf GAP domain is not drawn to scale. Abbreviations used are: ALPS, ArfGAP1 lipid-packing sensor domain; Ank, Ankyrin repeats; Arf GAP, Arf GTPase activating domain; BAR, Bin/Amphiphysin/Rvs domain; CALM, CALM binding domain; CB, clathrin box; CC, coiled-coiled domain; FG repeats, multiple copies of the XXFG motif; GLD, GTP-binding protein-like domain; PBS, paxillin binding site; PH, pleckstrin homology domain; Pro (PxxP)₃, cluster of three proline-rich (PxxP) motifs; Pro (D/ELPPKP)₈, eigth tandem Prolin-rich (D/ELPPKP) motifs; RA, Ras association motif; Rho GAP, Rho GTPase activating domain; SAM, sterile α-motif; SH3, Src homology 3 domain; SHD, Spa homology domain. *ASAP2 and ASAP3 lack the Pro (D/ELPPKP)₈ motifs. ASAP3 has no SH3 domain. ^&AGAP2 has a splice variant with three N-terminal PxxP motifs, called PIKE-L. @ARAP2 has an inactive Rho GAP domain.The subcellular localization and function of a number of Arf GAPs have been identified. Arf GAP1, Arf GAP2 and Arf GAP3 are found in the Golgi apparatus where they control membrane traffic by regulating Arf1•GTP levels.^12,13 Arf GAP1 has also been proposed to directly contribute to the formation of transport intermediates.¹⁴ SMAPs and AGAP1 and AGAP2 are associated with endosomes and regulate endocytic trafficking.^14,15 ASAPs, ARAPs and Gits are associated with FAs. ASAPs, ARAPs and ACAPs are found in actin-rich membrane ruffles. ASAP1 is also found in invadopodia and podosomes.⁴ We propose that common to all Arf GAPs is that they laterally organize membranes, which maintain surfaces of specialized functions such as FAs and podosomes/invadopodia. Some Arf GAPs function primarily as Arf effectors with the turnover rate of the specialized membrane surface being determined by the catalytic rate of the GAP. Other Arf GAPs function as Arf regulators that integrate several signals.ASAP1 is an example of an Arf GAP that may function as an Arf effector to regulate podosomes and invadopodia. ASAP1 is encoded by a gene on the short arm of chromosome 8. The gene is amplified in aggressive forms of uveal melanoma and cell migration rates correlate with ASAP1 expression levels in uveal melanoma¹⁶ and other cell types. ASAP1 function depends on cycling among four cellular locations, cytosol, FAs, lamellipodia and podosomes/invadopodia. ASAP1 is necessary for the formation of podosomes/invadopodia.^17,18The structural features of ASAP1 that are required to support podosome formation have been examined.^17,18 ASAP1 contains, from the N-terminus, BAR, PH, Arf GAP, Ank repeat, proline rich and SH3 domains (Fig. 2A).¹⁹ There are two major isoforms, ASAP1a and ASAP1b that differ in the proline rich domain. ASAP1a contains three SH3 binding motifs within the proline rich region including an atypical SH3 binding motif with 6 consecutive prolines. The atypical SH3 binding motif is absent in ASAP1b (Fig. 2A). ASAP1 also has a highly conserved tyrosine between the Ank repeat and proline rich domains that is a site of phosphorylation by the oncogene Src.¹⁸Open in a separate windowFigure 2ASAP1 function in podosome and invadopodia formation. (A) Domain structure of ASAP1 splice variants. ASAP1a contains three proline-rich motifs, P1, P2 and P3. P1 and P3 contain a typical (PxxP) motif. P2 contains six prolines. ASAP1b contains only P1 and P3. (B) Model of ASAP1 functioning as an Arf effector to regulate podosome and invadopodia formation. ASAP1 integrates signals from Src, PIP₂ and Arf•GTP. For abbreviations of the domain structure of ASAP1 see Figure 1. Other abbreviations: PIP₂, phosphoinositides 4,5-biphosphate; Arf1, ADP-ribosylation factor 1.The BAR domain is a bundle of 3 α-helices that homodimerizes to form a boomerang-shaped structure.^20,21 BAR domains sense or induce membrane curvature.²⁰ ASAP1 has been found to induce curvature dependent on its BAR domain.²² BAR domains are also protein binding sites.²¹ The BAR domain of ASAP1 binds to FIP3, a Rab11 and Arf6 binding proteins.²³ Arf6-dependent targeting of ASAP1 is likely mediated by FIP3.²³ Deletion of or introduction of point mutations into the BAR domain render ASAP1 inactive in supporting podosome formation. The relative role of membrane tubulation and protein binding in mediating the effect of the BAR domain on podosome formation has not been explored.The SH3 domain of ASAP1 binds to focal adhesion kinase²⁴ and pyk2.²⁵ Either deletion of or introduction of point mutations into the SH3 domain abrogates the ability of ASAP1 to support podosome formation.¹⁸ The molecular basis for the function of the SH3 domain in podosome formation is not known. The proline rich domain binds to Src¹⁹ and CrkL.²⁶ Whether it also binds to cortactin has not been resolved. Reports also conflict regarding the importance of the proline rich domain for podosomes/invadopodia formation.^17,18Three signals impinge on ASAP1 to drive podosome formation (Fig. 2B). A conserved tyrosine between the Ank and proline rich motifs is phosphorylated by Src.^18,25 Mutation of the tyrosine to phenylalanine results in a protein that functions as a dominant negative blocking podosome formation. ASAP1 with the tyrosine changed to glutamate can support podosome formation, but the mutant ASAP1 is not sufficient to drive podosome formation.¹⁸ Based on these results, phosphorylation of the conserved tyrosine is necessary but not sufficient to support podosome formation. Phosphatidylinositol 4,5-bisphosphate (PIP₂) binds to the PH domain, which stimulates GAP activity in vitro.²⁷ ASAP1 with mutations in the PH domain that abrogate binding, does not support podosome formation (Jian, Bharti and Randazzo PA, unpublished observations). Point mutations in the PH domain affect both the K_m and the k_cat for GAP activity. The effect of mutating the PH domain on the ability of ASAP1 to support podosome formation may be consequent to changes in binding Arf1•GTP; it is not likely the result of loss of GAP activity. ASAP1 with a point mutation in the GAP domain that prevents GAP activity but not Arf1•GTP binding is able to support podosome formation whereas a point mutant of ASAP1 that cannot bind Arf1•GTP does not (Jian, Bharti and Randazzo PA, unpublished observations).¹⁸ These data support the idea that ASAP1 integrates three signals, (1) PIP₂, (2) Src and (3) Arf1•GTP. In response to the signals, ASAP1 functions as a scaffold and directly alters the lipid bilayer to create a domain within the plasma membrane that becomes a podosome. In this model, ASAP1 is functioning as an Arf effector and the GAP activity may regulate the turnover of podosomes.ASAP3, another ASAP-type protein, is found in FAs.⁶ Reducing ASAP3 expression also reduces cell migration and invasion of Arf GAPs in cell migration mammary carcinoma cells through matrigel. Although ASAP3 does not affect the ability to form FAs, it does affect stress fiber formation and may affect focal adhesion maturation (Ha, Chen and Randazzo PA, unpublished observations).⁶ The molecular mechanisms underlying the effects of ASAP3 on the cytoskeleton are being examined including the possibility that, like ASAP1, ASAP3 integrates several signals and functions as an Arf effector.ARAPs are examples of Arf GAP family proteins that function as Arf regulators. In common with ASAPs, they integrate a number of signaling pathways and affect the actin cytoskeleton. Three genes encode ARAPs in humans.¹¹ Each of the ARAPs is comprised of a SAM, five PH, Arf GAP, Rho GAP, Ank repeat and Ras association domains. Two of the five PH domains have the consensus sequence for binding to the signaling lipid phosphoinositide 3,4,5-triphosphate (PIP₃); however, when examined for ARAP1, PIP₃ was not involved in membrane targeting (Campa F, Balla and Randazzo PA, unpublished observations).Examination of the role of ARAP2 in FA formation has provided information about the function of the GAP activity in the cellular function of an Arf GAP. ARAP2 selectively uses Arf6 as a substrate and, different from ARAP1 and ARAP3, has an inactive Rho GAP domain. The Rho GAP domain, however, retains the ability to selectively bind to RhoA•GTP. Also different from ARAP1 and ARAP3, ARAP2 associates with FAs. Cells with reduced expression of ARAP2, consequent to siRNA treatment, have fewer FAs and stress fibers and more focal complexes than control cells. The formation of FAs and stress fibers can be restored by expressing recombinant wild type ARAP2. A mutant of ARAP2 that lacks Arf GAP activity, while retaining the ability to bind to Arf6•GTP, cannot restore FA and stress fiber formation. Similarly, expression of a mutant of ARAP2 that is not able to bind RhoA•GTP cannot reverse the effect of reducing expression of endogenous ARAP2.²⁸ These results support the idea that ARAP2 functions as an Arf GAP that is an effector of RhoA.The model of ARAP2 functioning as a RhoA effector can explain the effects of ARAP2 on FAs (Fig. 3). Arf6•GTP is involved in the formation of Rac1•GTP.²⁹ Rac1•GTP drives lamellipodia and focal complex formation. The conversion of focal complexes to FAs is accompanied by an increase in RhoA•GTP and a decrease in Rac1•GTP. ARAP2 could function to mediate the reciprocal changes in RhoA and Rac1. RhoA•GTP formation leads to the activation of ARAP2. As a consequence of Arf6 GAP activity, Arf6•GTP is converted to Arf6•GDP. With reduced Arf6•GTP, Rac1•GTP concentration also decreases.Open in a separate windowFigure 3Model of ARAP2 as an Arf regulator that controls focal adhesion formation. In this model, ARAP2 functions as a RhoA effector. The inactive Rho GAP domain of ARAP2 binds to RhoA•GTP, which contributes to activation of Arf6 GAP activity. ARAP2 hydrolyzes its substrate Arf6•GTP into Arf6•GDP. Subsequent to Arf6•GTP hydrolysis, Rac1•GTP concentration decreases. For abbreviations of the domain structure of ARAP2 see Figure 1.The Arf GAP activity of other ARAPs may also be critical for cellular functions of the protein. Furthermore, the Rho GAP activity is slow for ARAP1 and ARAP3. It is possible that ARAP1 and ARAP3 can function as Rho effectors with an active Rho GAP domain analogously to ASAP1 functioning as an Arf effector. Further definition of the cellular function of ARAP1 and ARAP3 will provide opportunities to test this idea.We have provided two examples of Arf GAPs that affect cell adhesion and migration. In one case, the Arf GAP appears to function as an Arf effector. In the other case, the Arf GAP functions as a regulator of Arf. The difference in function was discerned using Arf GAP mutants. If functioning as an Arf effector, an Arf GAP mutant that can bind Arf•GTP but not induce hydrolysis can reverse the effect of reduced endogenous Arf GAP, whereas a mutant that cannot bind Arf•GTP cannot replace endogenous Arf GAP. When working as an Arf regulator, a mutant that can bind Arf•GTP but not induce GTP hydrolysis cannot replace endogenous Arf GAP. Whether functioning as an effector or regulator, the rate of GAP activity determines the turnover rate of a specialized membrane surface maintained by Arf.The Arf GAPs have specific sites of action within cells. Some contribute to malignancy, such as ASAP1, ASAP3, AGAP2 and SMAP1.³⁰ The molecular basis of cellular function of each Arf GAPs is distinct. Here, we describe one Arf GAP that functions as an Arf effector and another that functions as an Arf regulator. Each class of Arf GAP has distinct sets of protein binding partners. Furthermore, catalytic mechanism differs among the GAPs. Because of these differences, Arf GAPs may be useful therapeutic targets for cancer therapy.     

Arf GTPase-activating proteins and their potential role in cell migration and invasion

Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.     

TET family proteins and their role in stem cell differentiation and transformation

One of the main regulators of gene expression during embryogenesis and stem cell differentiation is DNA methylation. The recent identification of hydroxymethylcytosine (5hmC) as a novel epigenetic mark sparked an intense effort to characterize its specialized enzymatic machinery and to understand the biological significance of 5hmC. The recent discovery of recurrent deletions and somatic mutations in the TET gene family, which includes proteins that can hydroxylate methylcytosine (5mC), in a large fraction of myeloid malignancies further suggested a key role for dynamic DNA methylation changes in the regulation of stem cell differentiation and transformation.     

Helicoidal pattern in secondary cell walls and possible role of xylans in their construction

The helicoidal organization of secondary cell walls is overviewed from several examples. Both the plywood texture and the occurrence of characteristic defects strongly suggest that the wall ordering is relevant of a cholesteric liquid-crystal assembly that is rapidly and strongly consolidated by lignification. A preferential localization of glucuronoxylans, major matrix components, and in vitro re-association experiments emphasize their preeminent role: (1) during the construction of the composite as directing the cellulose microfibrils in a helicoidal array; (2) during the lignification of the composite as a host structure for lignin precursors.     

The role of human red blood cell membrane skeletal proteins in repetitive membrane strain and rupture

The influence of the membrane skeleton on cell membrane deformability, elasticity, and rupture after repetitive cycles of membrane strain, release and rupture was investigated. Human red blood cells were electrofused to doublets, which showed the post-fusion oscillation-movement. Geometrical developments of heat-treated cells were measured and compared to control cells. Alterations of cluster length and fusion zone diameter during repetitive colloidosmotic swelling period grow with heat treatment, and the number of precedent swell phases has minor influence on these values. Irrespective of the treatment, the geometrical doublet configuration at which a membrane rupture is initiated has an almost constant roundness index of 0.89. Increasing heat treatment temperature was shown to affect both deformability and elasticity of the membrane, such that doublets start each swell phase of the oscillation cycle from decreased roundness values. Evidence is given that there is a difference in the mechanical properties between the membrane at the fusion zone and the membrane of native red blood cells.