Annie Boned

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid‐dependent (microdomain model) or cytoskeleton‐based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin‐based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free‐like diffusion was observed when both the lipid‐dependent and cytoskeleton‐based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain

The murine neural cell adhesion molecule (NCAM) is known to exist in three isoforms of different size, NCAM‐180, −140 and −120 coded for by four transcripts of 6.9, 6.1, 4.8 and 2.7 kb in length. Since the differences between these isoforms are due to alternative splicing in the coding region for the transmembrane and cytoplasmic domains, the extracellular, N‐terminal portion of NCAM seemed to be shared by all three protein forms. Here we report that the coding region for N‐terminal domains of NCAM also contains at least two sites of alternative splicing, termed alpha and pi. Short additional sequences of 3, 18 and 30 nt in length can be introduced at these sites, which are located in the membrane‐proximal ‘stem’ between the Ig‐like domains and the membrane attachment site and within the Ig‐like domain IV, respectively. Proof for at least eight different mRNAs has been found by sequencing and S1 nuclease protection assays of selected independent cDNA clones, and Northern blot analyses. If most combination of the splice patterns identified so far in mouse brain occurred, 24 different mRNAs could be generated coding for 18 different proteins. The shortest extra‐sequence found inserted at splice site alpha consisted only of the trinucleotide AAG, raising questions about the mechanism of this particular insertion.

Transfected F3/F11 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.

Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt

In Drosophila the teashirt gene, coding for a zinc finger protein, is active in specific body parts for patterning. For example, Teashirt is required in the trunk (thorax and abdomen) tagmata of the embryo, parts of the intestine and the proximal parts of appendages. Here we report the isolation of vertebrate cDNAs related to teashirt. As in Drosophila, human and murine proteins possess three widely spaced zinc finger motifs. Additionally, we describe the expression patterns of the two murine genes. Both genes show regionalized patterns of expression, in the trunk, in the developing limbs and the gut.

High Degree of NCAM Diversity Generated by Alternative RNA Splicing in Brain and Muscle

The neural cell adhesion molecules (NCAMs) are cell surface glycoproteins involved in vertebrate cell contact formation. Several NCAM mRNA types are generated from a single primary transcript by alternative splicing and differential polyadenylation. In this presentation we analyse sequence heterogeneities within NCAM transcripts detected in the junctions of exons 7/8, 12/13 and 13/14. The highest degree of evolutionary conservation is observed in the 30‐nucleotide insertion (π) between exons 7 and 8, coding for an identical peptide sequence in the mouse, rat and chicken. The most complex splicing pattern is found between exons 12 and 13, called splice site a. Three alternative exons of 15, 48 and 42 nucleotides can be inserted in various combinations, which may also contain the additional trinucleotide AAG. In mouse muscle cell lines, differential ‘extra exon’ and AAG usage in splice site α creates up to 16 NCAM diversity forms, some (if not all) of which are also expressed in mouse brain. Additional microdiversity is generated by the insertion of an alternative AAG trinucleotide in exon junction 13/14. If all combinations of splicing patterns identified so far were to occur and to be translated, there could be up to 192 different NCAM proteins.

Dual-color fluorescence cross-correlation spectroscopy in a single nanoaperture: towards rapid multicomponent screening at high concentrations

Single nanometric apertures in a metallic film are used to develop a simple and robust setup for dual-color fluorescence cross-correlation spectroscopy (FCCS) at high concentrations. If the nanoaperture concept has already proven to be useful for single-species analysis, its extension to the dual-color case brings new interesting specificities. The alignment and overlap of the two excitation beams are greatly simplified. No confocal pinhole is used, relaxing the requirement for accurate correction of chromatic aberrations. Compared to two-photon excitation, nanoapertures have the advantage to work with standard fluorophore constructions having high absorption cross-section and well-known absorption/emission spectra. Thanks to the ultra-low volume analysed within one single aperture, fluorescence correlation analysis can be performed with single molecule resolution at micromolar concentrations, resulting in 3 orders of magnitude gain compared to conventional setups. As applications of this technique, we follow the kinetics of an enzymatic cleavage reaction at 2 muM DNA oligonucleotide concentration.We also demonstrate that FCCS in nanoaper-tures can be applied to the fast screening of a sample for dual-labeled species within 1 s acquisition time. This offers new possibilities for rapid screening applications in biotechnology at high concentrations.

Disruption of E2F signaling suppresses the INK4a-induced proliferative defect in M33-deficient mice

Polycomb group (Pc-G) proteins associate to form large complexes that repress Hox genes, thereby imposing Hox gene expression pattern required for development. However, Pc-G proteins have a Hox-independent function in controlling cell proliferation. Here we show that embryonic fibroblasts derived from M33-deficient mice are impaired in the progression into the S phase of the cell cycle, as shown by a reduced rate of incorporation of bromodeoxyuridine. These cells have a senescent phenotype, associated to an abnormal accumulation of the cyclin-dependent kinase inhibitor p16INK4a protein. We demonstrate that this defect is bypassed in mutant embryonic fibroblasts expressing a transdominant negative form of the cell cycle controlling transcription factor E2F (E2F-DB). In addition, we show that the polycomb protein M33 controls critical expansion of B- and T-lymphocyte precursors. Together, our results emphasize M33-Polycomb protein function in cell cycle control.

Genetic chasing of t helper cell idiotype and allotype genes.

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1+, 2.3−B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1+, 2.3−B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1b genes (B6) and not with Igh-1b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1+, 2.3−B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1+, 2.3−B6 T cells are produced by genes also linked to Igh-1b-allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.

On the molecular basis of T-helper-cell function: IV. B-lymphocyte-promotor factors: On their mode of action, biochemical nature and possible relationship to molecules involved in specific T-helper-cell activity

Some further aspects of B-lymphocyte-promotor factor (B-LPF) activity have been studied. This activity was present in the supernatants of certain helper-T-cell lines, and it induced polyclonal activation of Ig+ B cells into Ig-secreting cells. It was found that B-LPF induced polyclonal, terminal B-cell differentiation (1) in T-cell- and macrophage-depleted spleen cell populations, (2) in both Lyb-5- and Lyb-5+ cells as well as in small and blast-like splenic B cells, and (3) in normal rather than memory B cells. B-LPF function was neither restricted to major histocompatibility complex gene products nor to immunoglobulin allotypes. B-LPF-like activity was also produced by some B-cell lymphomas/hybrids and by the P388-D1 macrophage line. B-LPF activity was found in three MW fractions: (I) greater than 180,000 (pI greater than 7.0 and 4.5-5.5); (II) 50,000-70,000 (pI greater than 7.0; 6.0-6.5, and 4.5-5.5); and (III) 10,000-15,000 (pI greater than 7.0). All three MW forms of B-LPF activity carried antiserum 6036-defined and AB-1.9.3 monoclonal antibody-defined determinants, and they reacted with chicken gammaglobulin (CGG)-Sepharose but not with human serum albumin-Sepharose. These data indicate that the three MW forms of B-LPF activity are associated/dissociated forms of a 10,000-15,000 MW form (subunit) rather than three different molecular species with B-LPF activity. A comparative study between antigen-specific helper factors and B-LPF was hampered by the finding that the helper-T-cell hybridomas used (e.g., T85-109-45/1) only produced B-LPF in our hands. Previously, it has been described that these helper-T-cell hybrids produced CGG-specific, I-Ak-restricted helper factors. However, one surprising observation was that B-LPF produced by both T85 hybrid cells and L12 T lymphoma cells was absorbed and could be eluted from CGG-Sepharose columns. The relationship of B-LPF to other nonspecific and apparently specific T-helper-cell products is discussed in particular in the light of the observations that many immunologically active molecules are built up from 10,000-12,000 molecular weight domain-like polypeptide structures.

Electromicroelution and microfingerprinting of radiolabeled immunoglobulins from sodium dodecyl sulfate—polyacrylamide gels

An apparatus for electromicroelution from gel slices is described. After 20 min 70-90% of radiolabeled molecules can be extracted from the gel and recovered in 100 mul of buffer. The extracted molecules could be analysed in SDS-PAGE after reduction or could be split to perform microfingerprints (4 cm X 4 cm). This method has been used in several studies on membrane molecules (Fc gamma receptor, surface IgD, T cell receptor).