bluuebunny/crossref_metadata_2025 huggingface dataset:
Automation of a series of cutaneous topography measurements from silicon rubber replicas
Authors: Nardin P. , Nita D. , Mignot J.
Published: March 2003

Background/Aims Skin relief is a matter of interest for dermatologists and surgeons. One of the methods available for surface topography measurement is based on 3D profilometry using skin surface replicas. and most studies use statistical results obtained from a large number of skin replica samples.

The advent of optical profilometers (without contact) made it possible to remove the solid positive replica and to reduce the duration of the profilometric data acquisition. Nevertheless this saving of time, to be really interesting, needs to automate the data acquisition on a series of negative replicas.

Methods/Results By adding a video camera to the optical profilometer and then by processing the resulting images, we have conceived a system able to carry out topographic measurements on a series of replicas loosely organized on a sample holder, without any human intervention. The silicon replicas in use have a very light colour: nearly white, sometimes slightly blue or green. The laser spot of the profilometer is so luminous that its red colour looks white through the camera. When choosing a replica holder with a matt dark colour and marking the left upper corner of the study area on the replica in black ink, the colours to be differentiated on the image are then close to the black one and the white one.

We accordingly change the colour camera image into a black and white image (with 256 grey levels) and then carry out thresholdings to separate the different objects or information included in this image. With the use of a perfectly circular replica, of an accurately known size, laid on the sample holder at the center of the area filmed by the camera, we adjust the threshold level, which allows separation of the replica from its holder. We then move this calibrated replica in order to find the relationship between the size in pixels and the real size on the sample holder, in various positions of the video image.

The software has four main built‐in stages:

Moving the sample holder beneath the sensor until a part of a replica is detected in the field of view of the camera;

Moving the sample holder until this replica lies just in the middle of the image given by the camera;

Recognition of the mark of the upper left corner of the surface area to be measured out inside this replica; and

Moving the sample holder until the laser spot of the profilometer coincides with the origin of the surface area to be measured out, then carrying out this measurement.

From the upper left corner of the sample holder, a scanning, line‐by‐line or column‐by‐column (according to the selected priority direction), is carried out until the successive replicas are found, and is stopped as soon as the number of replicas entered by the operator is reached.

Conclusion The simplicity of the algorithms used makes it possible to distinguish the next measurement area from the preceding one in a few seconds.
Effect of exposure of human skin to a dry environment
Authors: Egawa Mariko , Oguri Motoki , Kuwahara Tomohiro , Takahashi Motoji
Published: March 2003

Background/aims: Changes in the skin conditions after exposure to low humidity have been generally experienced in everyday life, but there have been few reports to approach it—especially in healthy skin. We have examined the effect of low humidity on healthy human skin by using noninvasive measurement devices.

Methods: Skin conditions on the ventral forearm and the cheek before and after 3 or 6 h exposure to low humidity were evaluated by measuring skin surface conductance, skin surface capacitance and transepidermal water loss. Skin surface replicas were also taken before and after exposure and analysed for roughness parameters—Ra (arithmetic mean roughness value), Rz (10‐point height), Sm (mean value of the profile element) and VC1 (anisotropy of skin furrows).

Results: There was a significant decrease of water content of stratum corneum at both test sites from the time points 0 h to 3 h and 6 h (P < 0.01) and transepidermal water loss from the time point 0 h to 6 h (P < 0.05). Regarding the roughness parameters, a significant increase of Rz in the directions of 45°/225° and 90°/270° to the body axis and Sm in the directions of 0°/180° (P < 0.05) on the forearm and VC1 (P < 0.05) on the cheek. The parameter Rz also showed a tendency to increase in the directions of 45°/225° (P = 0.06) on the cheek. A specific pattern of the changes to be related to the Langer's lines in the surface morphology was observed. The changes of skin surface pattern in our experiment lead us to consider that exposure to low humidity even in such a short period would be related to inducing aggravation of skin texture and the formation of fine wrinkles.

Conclusion: A short exposure of skin to a low‐humidity environment induced changes in the moisture contents in the stratum corneum and skin surface pattern, which lead us to assume that a dry environment in our daily life would make fine wrinkles related to lack of water in the stratum corneum.
The Yeast Endosomal t‐SNARE, Pep12p, Functions in the Absence of its Transmembrane Domain
Authors: Gerrard Sonja R. , Mecklem Alison B. , Stevens Tom H.
Published: March 2003

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t‐SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar protein transport. Our screen for temperature‐sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non‐permissive temperature. Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain. Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion. In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step. Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes.
Facing Inward from Compartment Shores: How Many Pathways were we Looking For?
Authors: Johannes Ludger , Goud Bruno
Published: March 2003

Protein toxins of the Shiga family have become potent tools in studying a number of intracellular transport events such as endocytosis, the communication between endosomes and the biosynthetic/secretory pathway, and retrograde transport from the Golgi apparatus to the endoplasmic reticulum. It seems clear today that most of these transport events can be explained from the toxins’ interactions with cellular factors. This review will primarily focus on the discussion of recent data obtained on Shiga toxin and related toxins. We will point out to what extent the study of these proteins has opened new avenues for the development of intracellular targeting tools.
Antigen Traffic Pathways in Dendritic Cells
Authors: Watts Colin , Amigorena Sebastian
Published: March 2003

Dendritic cells (DC) are now believed to be the principal initiators of T cell‐mediated immune responses. Their location in body tissues, migratory behaviour in response to inflammatory stimuli, endocytic properties, expression of MHC molecules and key T cell stimulatory molecules and many other attributes place these remarkable cells in a unique and influential position in the immune system. Progress in DC culture methods has recently allowed in‐depth studies on the cell biological features that enable them to fulfil their crucial role in the immune response.
Role of Cholesterol in Developing T‐Tubules: Analogous Mechanisms for T‐Tubule and Caveolae Biogenesis
Authors: Carozzi Amanda J. , Ikonen Elina , Lindsay Margaret R. , Parton Robert G.
Published: March 2003

Recent work has suggested that caveolae biogenesis and transverse‐tubule (T‐tubule) formation in muscle cells share similar underlying features. We compared the properties of caveolin‐1 (cav‐1)‐positive caveolae, in epithelial cells, with caveolin‐3 (cav‐3)‐positive precursor T‐tubules, in differentiating C2C12 muscle cells, using the cholesterol‐binding drug, Amphotericin B (AmphB). Treatment of MDCK epithelial cells with acute high doses or chronic low doses of AmphB caused a loss of surface caveolae and the rapid redistribution of cav‐1, and exogenously expressed cav‐3, from the cell surface into modified endosomes. This effect was reversible and specific, as the GPI‐anchored protein, alkaline phosphatase, was largely unaffected by the treatment unless it had been previously partitioned into caveolar domains. In differentiating C2C12 mouse myotubes, AmphB also caused a complete redistribution of cav‐3 from precursor T‐tubule elements into enlarged endosomes, morphologically very similar to those seen in MDCK cells. This was accompanied by redistribution of a T‐tubule marker and a dramatic reduction in the extent of surface‐connected tubular elements. We propose that cholesterol‐enriched glycolipid ‘raft’ domains are involved in the formation and maintenance of diverse membrane systems including caveolae and the T‐tubule system of muscle.
A Functional Role for VAP‐33 in Insulin‐Stimulated GLUT4 Traffic
Authors: Foster Leonard J. , Weir M. Lynn , Lim Dawn Y. , Liu Zhi , Trimble William S. , Klip Amira
Published: March 2003

Soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle‐associated membrane protein‐2 (VAMP‐2) is a SNARE protein involved in insulin‐dependent glucose transporter 4 (GLUT4) traffic. VAMP‐2 is required for productive GLUT4 incorporation into the plasma membrane. VAMP‐associated protein of 33 kDa (VAP‐33) is an integral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin‐dependent traffic of GLUT4, we show that VAP‐33 colocalized significantly with VAMP‐2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild‐type VAP‐33 in L6 myoblasts attenuated the insulin‐dependent incorporation of myc‐tagged GLUT4 into the plasma membrane, and this response was restored by co‐overexpression of VAMP‐2 linked to green fluorescent protein. Antibodies to VAP‐33 microinjected into 3T3‐L1 adipocytes abrogated the insulin‐stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP‐2‐containing compartments from L6 myotubes and 3T3‐L1 adipocytes showed significant levels of VAP‐33. We propose that VAP‐33 may be a regulator of VAMP‐2 availability for GLUT4 traffic and other vesicle fusion events.
Endocytic Recycling is Required for the Presentation of an Exogenous Peptide via MHC Class II Molecules
Authors: Pathak Sulabha S. , Blum Janice S.
Published: March 2003

Exogenous antigenic peptides captured and presented in the context of major histocompatibility (MHC) class II molecules on APC, have been employed as potent vaccine reagents capable of activating cellular immune responses. Binding and presentation of select peptide via surface class II molecules has been reported. Here, a role for endocytosis and early endosomes in the presentation of exogenous peptides via MHC class II molecules is described. T cell recognition of a 14 amino acid human serum albumin‐derived peptide in the context of HLA‐DR4 was observed only with metabolically active APC. The delayed kinetics and temperature dependence of functional peptide presentation via APC, were consistent with a requirement for peptide internalization to early endosomal compartments prior to T cell recognition. Ablating endocytosis by exposing cells to inhibitors of ATP production completely blocked the display of functional peptide:class II complexes on the surface of the APC. Presentation of the peptide was also found to be sensitive to primaquine, a drug that perturbs the recycling of transport vesicles containing endocytic receptors and mature class II complexes. Functional presentation of the endocytosed peptide was dependent upon these mature class II complexes, as inhibitor studies ruled out a requirement for newly synthesized class II molecules. N‐terminal processing of the endocytosed peptide was observed upon trafficking through endosomal compartments and linked to the formation of functional peptide:class II complexes. These findings establish a novel mechanism for regulating class II‐restricted peptide presentation via the endocytic pathway.
The Simian Immunodeficiency Virus Envelope Glycoprotein Contains Multiple Signals that Regulate its Cell Surface Expression and Endocytosis
Authors: Bowers Katherine , Pelchen‐Matthews Annegret , Höning Stefan , Vance Patricia J. , Creary Lisa , Haggarty Beth S. , Romano Josephine , Ballensiefen Wolfgang , Hoxie James A. , Marsh Mark
Published: March 2003

The cell surface expression of the envelope glycoproteins (Envs) of primate immunodeficiency viruses is, at least in part, regulated by endocytosis signal(s) located in the Env cytoplasmic domain. Here, we show that a membrane proximal signal that directs the simian immunodeficiency virus (SIV) Env to clathrin‐coated pits, and is conserved in all SIV and human immunodeficiency virus Envs, conforms to a Yxxø motif (where x can be any amino acid and Ø represents a large hydrophobic residue). This motif is similar to that described for a number of cellular membrane proteins. By surface plasmon resonance we detected a high affinity interaction between peptides containing this membrane proximal signal and both AP1 and AP2 clathrin adaptor complexes. Mutation of the tyrosine in this membrane proximal motif in a SIV Env with a prematurely truncated cytoplasmic domain leads to a ≥25‐fold increase in Env expression on infected cells. By contrast, the same mutation in an Env with a full‐length cytoplasmic domain increases cell surface expression only 4‐fold. We show that this effect results from the presence of additional endocytosis signals in the full‐length cytoplasmic domain. Chimeras containing CD4 ecto‐ and membrane spanning domains and a full‐length SIV Env cytoplasmic domain showed rapid endocytosis even when the membrane proximal tyrosine‐based signal was disrupted. Mapping experiments indicated that at least some of the additional endocytosis information is located between residues 743 and 812 of Env from the SIVmac239 molecular clone. Together, our findings indicate that the cytoplasmic domain of SIV Env contains multiple endocytosis and/or trafficking signals that modulate its surface expression on infected cells, and suggest an important role for this function in pathogenesis.
Proteomic Analysis of Two Functional States of the Golgi Complex in Mammary Epithelial Cells
Authors: Wu Christine C. , Yates John R. , Neville Margaret C. , Howell Kathryn E.
Published: March 2003

Organellar compartments involved in secretion are expanded during the transition from late pregnancy (basal secretory state) to lactation (maximal secretory state) to accommodate for the increased secretory function required for copious milk production in mammary epithelial cells. The Golgi complex is a major organelle of the secretory pathway and functions to sort, package, distribute, and post‐translationally modify newly synthesized proteins and membrane lipids. These complex functions of the Golgi are reflected in the protein complement of the organelle. Therefore, using proteomics, the protein complements of Golgi fractions isolated at two functional states (basal and maximal) were compared to identify some of the molecular changes that occur during this transition. This global analysis has revealed that only a subset of the total proteins is up‐regulated from steady state during the transition. Identification of these proteins by tandem mass spectrometry has revealed several classes of proteins involved in the regulation of membrane fusion and secretion. This first installment of the functional proteomic analysis of the Golgi complex begins to define the molecular basis for the transition from basal to maximal secretion.
Weibel‐Palade Body Membrane Proteins Exhibit Differential Trafficking After Exocytosis in Endothelial Cells
Authors: Arribas Monica , Cutler Daniel F.
Published: March 2003

Weibel‐Palade bodies, the secretory granules of endothelial cells, possess two different membrane proteins. However, P‐selectin is seen only in Weibel‐Palade bodies in HUVECs, whereas CD63 is also seen in late endosomes/lysosomes. Since P‐selectin is targeted to lysosomes in heterologous expression studies, we have determined whether a lysosomal targeting signal also operates within HUVECs. We have also examined the trafficking of CD63 to its two different intracellular locations. By following antibodies bound at the plasma membrane during stimulation, we have discovered that while half of the P‐selectin recycles to the WPBs, 50% is rapidly delivered to a lamp‐1‐positive compartment. Thus, the lysosomal targeting signal of this protein also operates in HUVECs. CD63 is found constitutively at the cell surface of HUVECs and most of it is delivered to the late endosomes/lysosomes after internalisation. However, stimulation causes both a rise in the CD63 plasma membrane level and in the amount that recycles to the WPBs. Our data strongly suggest that the CD63 that originates in the WPB preferentially recycles to the granule rather than being delivered to the late endosome/lysosome, and that there are, therefore, two separate pools of this protein within HUVECs. Our findings indicate that although P‐selectin and CD63 are both targeted to the same compartments from the PM, the kinetics and the ratio of their targeting to Weibel‐Palade bodies versus lysosomes are very different.
Detergent Insoluble Microdomains are not Involved in Transcytosis of Polymeric Ig Receptor in FRT and MDCK Cells
Authors: Sarnataro Daniela , Nitsch Lucio , Hunziker Walter , Zurzolo Chiara
Published: March 2003

In polarized epithelial cells, sorting of proteins and lipids to the apical or basolateral domain of the plasma membrane can occur via direct or indirect (transcytotic) pathways from the trans Golgi network (TGN). The ‘rafts’ hypothesis postulates that the key event for direct apical sorting of some transmembrane proteins and the majority of GPI‐anchored proteins depends on their association with glycosphingolipid and cholesterol enriched microdomains (rafts). However, the mechanism of indirect sorting to the apical membrane is not clear. The polyimmunoglobulin receptor (pIgR) is one of the best studied proteins that follow the transcytotic pathway. It is normally delivered from the TGN to the basolateral surface of polarized Madin–Darby Canine Kidney (MDCK) cells from where it transports dIgA or dIgM to the apical surface. We have studied the intracellular trafficking of pIgR in Fischer rat thyroid cells (FRT), and have investigated the sorting machinery involved in transcytosis of this receptor in both FRT and MDCK cells. We found that, in contrast with MDCK cells, a significant amount (∼30%) of pIgR reaches the apical surface by a direct pathway. Furthermore, in both cell lines it does not associate with Triton X‐100‐insoluble microdomains, suggesting that at least in these cells ‘rafts’ are not involved in basolateral to apical transcytosis.
The GRIP Domain is a Specific Targeting Sequence for a Population of trans‐Golgi Network Derived Tubulo‐Vesicular Carriers
Authors: Brown Darren  L. , Heimann Kirsten , Lock John , Kjer‐Nielsen Lars , van Vliet Catherine , Stow Jennifer L. , Gleeson Paul A.
Published: March 2003

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled‐coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno‐electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)‐p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans‐Golgi network (TGN). Real‐time imaging of live HeLa cells revealed that the GFP‐p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP‐p230GRIP‐transfected cells. Analysis of Golgi‐derived vesicles by sucrose gradient fractionation demonstrated that GFP‐p230GRIP binds to a specific population of vesicles distinct from those labelled for β‐COP or γ‐adaptin. The GFP‐p230GRIP fusion protein is recruited to the same vesicle population as full‐length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans‐Golgi network tubulo‐vesicular carriers.
A Third Specificity‐Determining Site in μ2 Adaptin for Sequences Upstream of YxxΦ Sorting Motifs
Authors: Owen David J. , Setiadi Hendra , Evans Philip R. , McEver Rodger P. , Green Samuel A.
Published: March 2003

Internalization signals of the YxxΦ type (Φ=bulky hydrophobic side chain) interact with the μ2 chain of AP‐2 adaptors. Internalization activity is intolerant of non‐conservative substitution of either the tyrosine or the Φ side chains, which bind to hydrophobic pockets in μ2 adaptin in a conformation described as ‘a two pinned plug into a socket’. P‐selectin, a type I transmembrane protein, contains the YxxΦ‐like sequence YGVF in its cytoplasmic domain, but substitution of either the tyrosine or phenylalanine with alanine in the full‐length protein causes only small changes in the rate of endocytosis. It is shown here that the sequence YGVF contained within a peptide corresponding to the 17 COOH‐terminal amino acids of P‐selectin binds to μ2 adaptin in the same fashion previously seen for other YxxΦ motifs. In addition, the P‐selectin peptide binds to a third hydrophobic pocket in μ2 adaptin through a leucine at position Y−3 in the peptide. This structure suggests that some sequences can function as a ‘three pinned plug’, in which internalization activity is not critically dependent on any one of the three interacting side chains.
Secretory and Endocytic Pathways Converge in a Dynamic Endosomal System in a Primitive Protozoan
Authors: Ghedin Elodie , Debrabant Alain , Engel Juan C. , Dwyer Dennis M.
Published: March 2003

Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania. This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface‐expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3′nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid‐phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.
Cytoplasmic Signals Mediate Apical Early Endosomal Targeting of Endotubin in MDCK Cells
Authors: Gokay K. E. , Young R. S. , Wilson J. M.
Published: March 2003

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N‐glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid‐rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac‐antigen chimeras and expressed them in Madin–Darby canine kidney cells. We show that both the apically targeting wild‐type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX‐100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.
Approaching the Molecular Mechanism of Autophagy
Authors: Stromhaug Per E. , Klionsky Daniel J.
Published: March 2003

Autophagy is a complex cellular process that involves dynamic membrane rearrangements under a range of physiological conditions. It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases. Upon induction of autophagy, cytoplasm is sequestered into vesicles and delivered to a degradative organelle, the vacuole in yeast or the lysosome in mammalian cells. The process is unique in that it converts material that is topologically intracellular into topologically extracellular. Autophagy was first described more than 50 years ago, but it is since the discovery of the pathway in yeast cells that our knowledge about the molecular events taking place during the process has expanded. The generation of autophagy‐specific mutants in a variety of yeast cell lines has provided insight into functional roles of more than 15 novel genes, double that number if we include genes whose products function also in other processes. Although we have learned much about autophagy, many questions remain to be answered. This review highlights the most recent advances in the autophagy field in both yeast and mammalian cells.
Selective Control of Basolateral Membrane Protein Polarity by Cdc42
Authors: Cohen David , Müsch Anne , Rodriguez‐Boulan Enrique
Published: March 2003

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase‐deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase‐deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine‐tune their membrane composition in response to extracellular signals during development, migration and in response to injury.
Deconstructing Golgi Inheritance
Authors: Rossanese Olivia W. , Glick Benjamin S.
Published: March 2003

Eukaryotic cells use a variety of strategies to inherit the Golgi apparatus. During vertebrate mitosis, the Golgi reorganizes dramatically in a process that seems to be driven by the reversible fragmentation of existing Golgi structures and the temporary redistribution of Golgi components to the endoplasmic reticulum. Several proteins that participate in vertebrate Golgi inheritance have been identified, but their detailed functions remain unknown. A comparison between vertebrates and other eukaryotes reveals common mechanisms of Golgi inheritance. In many cell types, Golgi stacks undergo fission early in mitosis. Some cells exhibit a further Golgi breakdown that is probably due to a mitotic inhibition of membrane traffic. In all eukaryotes examined, Golgi inheritance involves either the partitioning of pre‐existing Golgi elements between the daughter cells or the emergence of new Golgi structures from the endoplasmic reticulum, or some combination of these two pathways.
Elevated Endosomal pH in HeLa Cells Overexpressing Mutant Dynamin Can Affect Infection by pH‐Sensitive Viruses
Authors: Huber Marlis , Brabec Marianne , Bayer Nora , Blaas Dieter , Fuchs Renate
Published: March 2003

Many viruses gain access to the cell via the endosomal route and require low endosomal pH for infectivity. The GTPase dynamin is essential for clathrin‐dependent endocytosis, and in HeLa cells overexpressing the nonfunctional dynaminK44A mutant the formation of clathrin‐coated vesicles is halted. HRV2, a human minor group rhinovirus, is internalized by members of the low‐density lipoprotein receptor family in a clathrin‐independent manner. The low endosomal pH then leads to conversion of the capsid to C‐antigen, which is required for release (uncoating) and transfer of the viral RNA into the cytosol and de novo synthesis of infectious virus. We here demonstrate that overexpression of dynaminK44A reduces this antigenic conversion and results in diminished viral synthesis. In contrast, lysosomal degradation is unaffected. The kinetics of the formation of C‐antigen in vitro and in vivo suggest that the pH in endosomes is elevated by about 0.4 units upon overexpression of dynaminK44A. As a consequence, HRV2 uncoating is diminished early after internalization but attains control levels upon prolonged internalization. Thus, overexpression of dynaminK44A, in addition to trafficking defects, results in an elevated endosomal pH and thereby affects virus infection and most likely endosomal sorting and processing.
Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells
Authors: Elson Elliot L.
Published: March 2003

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross‐correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.
Near‐Field Scanning Optical Microscopy, a Siren Call to Biology
Authors: Edidin Michael
Published: March 2003

Near‐field illumination of a sample with visible light can resolve features well beyond the resolution of conventional, far‐field microscopes. Near‐field scanning optical microscopy (NSOM) then has the potential of extending the resolution of techniques such as fluorescent labeling, yielding images of cell structures and molecules on the nanoscale. However, major problems remain to be solved before NSOM can be easily used for wet biological samples. The most significant of these is control of the distance between near‐field aperture and the sample surface. Hence, while NSOM promises much, its application to biology is about where electron microscopy was 40 or 50 years ago.
Drosophila Amphiphysin is a Post‐Synaptic Protein Required for Normal Locomotion but Not Endocytosis
Authors: A. Leventis Peter , M. Chow Brenda , Stewart Bryan A. , Iyengar Balaji , Campos Ana Regina , Boulianne Gabrielle L.
Published: March 2003

Clathrin‐mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin‐mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.
Selective Delivery of Secretory Cargo in Golgi‐Derived Carriers of Nonepithelial Cells
Authors: Rustom Amin , Bajohrs Mark , Kaether Christoph , Keller Patrick , Toomre Derek , Corbeil Denis , Gerdes Hans‐Hermann
Published: March 2003

In epithelial cells, soluble cargo proteins destined for basolateral or apical secretion are packaged into distinct trans‐Golgi network‐derived transport carriers. Similar carriers, termed basolateral‐ and apical‐like, have been observed in nonepithelial cells using ectopically expressed membrane marker proteins. Whether these cells are capable of selectively packaging secretory proteins into distinct carriers is still an open question. Here, we have addressed this issue by analyzing the packaging and transport of secretory human chromogranin B fusion proteins using a green fluorescent protein‐based high‐resolution, dual‐color imaging technique. We were able to show that these secretory markers were selectively packaged at the Golgi into tubular/vesicular‐like transport carriers containing basolateral membrane markers, resulting in extensive cotransport. In contrast, deletion mutants of the human chromogranin B fusion proteins lacking an N‐terminal loop structure were efficiently transported in both basolateral‐ and apical‐like carriers, the latter displaying a spherical morphology. Similarly, in polarized epithelial cells, the human chromogranin B fusion protein was secreted basolaterally and the loop‐deleted analogue into both the basolateral and apical medium. These findings suggest that nonepithelial cells, like their epithelial counterparts, possess a sorting machinery capable of selective packaging of secretory cargo into distinct types of carriers.
EHD3: A Protein That Resides in Recycling Tubular and Vesicular Membrane Structures and Interacts with EHD1
Authors: Galperin Emilia , Benjamin Sigi , Rapaport Debora , Rotem‐Yehudar Rinat , Tolchinsky Sandra , Horowitz Mia
Published: March 2003

Here we report the characterization of an eps15 homology (EH) domain containing protein designated EHD3. EHD3 was mapped to human chromosome 2p22–23, while the murine Ehd3 homolog was mapped to chromosome 17p21. Both the human and the mouse genes contain a polymorphic (CA) repeat in their 3′UTR. One 3.6‐kb Ehd3 transcript was mainly detected in adult mouse brain and kidney and at day 7 of mouse development. On the other hand, human tissues exhibited two, 4.2‐ and 3.6‐kb, EHD3 RNA species. They were predominantly expressed in heart, brain, placenta, liver, kidney and ovary. EHD3, expressed as a green fluorescent fusion protein was localized to endocytic vesicles and to microtubule‐dependent, membrane tubules. There was a clear colocalization of EHD3‐positive structures and transferrin‐containing recycling vesicles, implying that EHD3 resides within the endocytic recycling compartment. Shuffling the N‐terminal domain of EHD1 (previously shown to reside in the transferrin‐containing, endocytic recycling compartment) with that of EHD3 resulted in a chimeric EHD protein that was localized mainly to tubules instead of the endocytic vesicles, implicating the N‐terminal domain as responsible for the tubular localization of EHD3. Mutant EHD3 forms, missing the N‐terminal or the C‐terminal domains, lost their tubular localization. Results of two‐hybrid analyses indicated that EHD1 and EHD3 interact with each other. In addition, EHD1 and EHD3 could be coimmunoprecipitated from cellular extracts, confirming the interaction implied by two‐hybrid analysis. Moreover, coexpression of EHD1 and EHD3 resulted in their colocalization in microtubule‐dependent tubules as well as in punctate forms. Based on its specific intracellular localization and its interaction with EHD1, we postulate that EHD3 localizes on endocytic tubular and vesicular structures and regulates their microtubule‐dependent movement.
The μ2 Subunit of the Clathrin Adaptor AP‐2 Binds to FDNPVY and YppØ Sorting Signals at Distinct Sites
Authors: Boll Werner , Rapoport Iris , Brunner Christian , Modis Yorgo , Prehn Siegfried , Kirchhausen Tomas
Published: March 2003

The endocytic sorting signal on the low‐density lipoprotein receptor for clathrin‐mediated internalization is the sequence FDNPVY in the receptor's cytosolic tail. We have used a combination of surface plasmon resonance and crosslinking with a photoactivated peptide probe to demonstrate the interaction between FDNPVY‐containing peptides and the μ2 chain of purified AP‐2 clathrin adaptors (the complexes responsible for plasma membrane sorting). We show that recognition of the FDNPVY signal is mediated by a binding site in the μ2‐subunit that is distinct from the site for the more general YppØ sorting signal, another tyrosine‐based sequence also recognized by μ2‐adaptin. These results suggest the possibility that low‐density lipoprotein receptor uptake may be modulated specifically and independently of other proteins in the clathrin pathway.
Identification of RanBP2‐ and Kinesin‐Mediated Transport Pathways with Restricted Neuronal and Subcellular Localization
Authors: Mavlyutov Timur A. , Cai Yunfei , Ferreira Paulo A.
Published: March 2003

Ran‐binding proteins, karyopherins, and RanGTPase mediate and impart directionality to nucleocytoplasmic transport processes. This biological process remains elusive in neurons. RanBP2 has been localized at the nuclear pore complexes and is very abundant in the neuroretina. RanBP2 mediates the assembly of a large complex comprising RanGTPase, CRM1/exportin‐1, importin‐β, KIF5‐motor proteins, components of the 19S cap of the 26S proteasome, ubc9 and opsin. Here, we show RanBP2 is abundant in the ellipsoid compartment of photoreceptors and RanGTPase‐positive particles in cytoplasmic tracks extending away from the nuclear envelope of subpopulations of ganglion cells, suggesting RanBP2's release from nuclear pore complexes. KIF5C and KIF5B are specifically expressed in a subset of neuroretinal cells and differentially localize with RanBP2 and importin‐β in distinct compartments. The C‐terminal domains of KIF5B and KIF5C, but not KIF5A, associate directly with importin‐β in a RanGTPase‐dependent fashion in vivo and in vitro, indicating importin‐β is an endogenous cargo for a subset of KIF5s in retinal neurons. The KIF5 transport pathway is absent from the myoid region of a topographically distinct subclass of blue cones and the distribution of kinesin‐light chains is largely distinct from its KIF5 partners. Altogether, the results identify the existence of neuronal‐ and subtype‐specific kinesin‐mediated transport pathways of importin‐β‐bound cargoes to and/or from RanBP2 and indicate RanBP2 itself may also constitute a scaffold carrier for some of its associated partners. The implications of these findings in protein kinesis and pathogenesis of degenerative neuropathies are discussed.
SopD2 is a Novel Type III Secreted Effector of Salmonella typhimurium That Targets Late Endocytic Compartments Upon Delivery Into Host Cells
Authors: Brumell John H. , Kujat‐Choy Sonya , Brown Nat F. , Vallance Bruce A. , Knodler Leigh A. , Finlay B. Brett
Published: March 2003

Salmonella typhimuriumis a facultative intracellular pathogen that utilizes two type III secretion systems to deliver virulence proteins into host cells. These proteins, termed effectors, alter host cell function to allow invasion into and intracellular survival/replication within a vacuolar compartment. Here we describe SopD2, a novel member of the Salmonella translocated effector (STE) family, which share a conserved N‐terminal type III secretion signal. Disruption of the sopD2 gene prolonged the survival of mice infected with a lethal dose of Salmonella typhimurium, demonstrating a significant role for this effector in pathogenesis. Expression of sopD2 was induced inside host cells and was dependent on functional ssrA/B and phoP/Q, two component regulatory systems. HA‐tagged SopD2 was delivered into HeLa cells in a SPI‐2‐dependent manner and associated with both the Salmonella‐containing vacuole and with swollen endosomes elsewhere in the cell. Subcellular fractionation confirmed that SopD2 was membrane associated in host cells, while the closely related effector SopD was localized to the cytosol. A SopD2 fusion to GFP associated with small tubular structures and large vesicles containing late endocytic markers, including Rab7. Surprisingly, expression of N‐terminal amino acids 1–150 of SopD2 fused to GFP was sufficient to mediate both binding to late endosomes/lysosomes and swelling of these compartments. These findings demonstrate that the N‐terminus of SopD2 is a bifunctional domain required for both type III secretion out of Salmonella as well as late endosome/lysosome targeting following translocation into host cells.
Raft Partitioning of the Yeast Uracil Permease During Trafficking Along the Endocytic Pathway
Authors: Dupré Sophie , Haguenauer‐Tsapis Rosine
Published: March 2003

Lipid rafts, formed by the lateral association of sphingolipids and cholesterol in the external membrane leaflet, have been implicated in membrane traffic and cell signaling in mammalian cells. Yeast plasma membranes were also recently shown to contain lipid raft microdomains consisting of sphingolipids and ergosterol, and containing several plasma membrane proteins, including Gas1p, a GPI‐anchored protein, and the [H+] ATPase Pma1p. In this study, we investigated whether lipid rafts were involved in the intracellular trafficking of a yeast transporter, uracil permease, which undergoes ubiquitin‐dependent endocytosis. Regardless of its ubiquitination status, uracil permease was found to be associated with rafts in the plasma membrane. The expression of Fur4p in lcb1–100 cells, deficient in the first enzyme of sphingolipid synthesis, impaired the association of Fur4p with detergent‐resistant fractions. When targeted to endocytic compartments, uracil permease appeared to be progressively transferred to detergent‐soluble fractions, suggesting that the lipid environment might change between plasma membrane and endosomes. Consistent with this hypothesis, the wild‐type form of the v‐SNARE Snc1p, which is known to cycle between the plasma membrane and endosomal compartments, was recovered in both detergent‐resistant and detergent‐soluble fractions. In contrast, a variant Snc1p that accumulates at the plasma membrane was recovered exclusively in detergent‐resistant fractions.
Immunity to Candida
Authors: Fidel PL
Published: March 2003

Candida
species are commensal fungal organisms as well as opportunistic pathogens of mucosal tissues. From the commensal relationship, most healthy individuals have demonstrable
Candida
‐specific immunity. In immunocompromised persons, however, fungal infections caused primarily by
C. albicans
often occur. In HIV disease, up to 90% of HIV+ persons will have a symptomatic episode of oropharyngeal candidiasis (OPC) sometime during progression to AIDS, many of which become recurrent. In contrast, vulvovaginal candidiasis (VVC) and systemic
Candida
infections (candidaemia) are much less common during HIV disease, indicating the diversity and compartmentalization of the host response to
Candida
. Both innate resistance and acquired immunity play some role in maintaining
C. albicans
in the commensal state and protecting the systemic circulation. Polymorphonuclear leukocytes (PMNL) are critical for protection against systemic infections, whereas cell‐mediated immunity (CMI) by Th1‐type CD4
+
T‐cells is important for protection against mucosal infections. However, there is a discordant role for CMI at the vaginal versus oral mucosa, whereas little to no role for local or systemic CMI is evident at the vaginal mucosa. In contrast, there is a strong correlation between reduced blood CD4
+
cells and the incidence of OPC, but it remains unclear whether systemic or local CMI is more important. Evaluation of systemic CMI in a cohort of HIV+ individuals with and without mucosal candidiasis revealed that
Candida
‐specific CMI is not different between HIV+ persons with OPC or VVC and HIV– persons. Thus, the correlation of reduced CD4
+
cell numbers to OPC may be explained by the requirement for a threshold number of systemic CD4
+
cells to protect the oral mucosa together with the status of local immunity. Indeed, HIV+ persons with and without OPC had a Th2‐type salivary cytokine profile suggestive of susceptibility to
Candida
infection compared with a protective Th0/Th1‐type profile in HIV– persons.
Candida
‐specific antibodies, although present, are controversial relative to a role in protection or eradication of infection. While studies of mucosal innate resistance are limited, we recently found that epithelial cells from saliva and vaginal lavages of healthy individuals inhibit the growth of
Candida in vitro
. This epithelial cell anti‐
Candida
activity requires cell contact by viable cells with no role for soluble factors, including saliva. Interestingly, oral epithelial cells from HIV+ persons with OPC had significantly reduced activity, indicating some protective role for the epithelial cells. Taken together, these data suggest that immunity to
Candida is site‐specific, compartmentalized and involves innate and/or acquired mechanisms from systemic and/or local sources.