Force Measurements of the α5β1 Integrin–Fibronectin Interaction

The interaction of the α₅β₁ integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α₅β₁/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α₅β₁ and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α₅β₁ expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α₅β₁/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α₅β₁/FN7-10 interactions. The dynamic rupture force of the α₅β₁/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α₅β₁/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.     

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ⁺CD8αα⁺ IELs. In the absence of Kdm6b, TCRαβ⁺CD8αα⁺ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ⁺CD8αα⁺ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ⁺CD8αα⁺ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ⁺CD8αα⁺ IELs.Subject terms: Epigenetics, Gene regulation, Immunological disorders, T cells     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

Function of the Tetraspanin CD151–α6β1 Integrin Complex during Cellular Morphogenesis

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.     

An ancient interleukin-16–like molecule regulates hemocyte proliferation via integrin β1 in invertebrates

Interleukins (ILs) are cytokines with crucial functions in innate and adaptive immunity. IL genes are only found in vertebrates, except for IL-16, which has been cloned in some arthropod species. However, the function of this gene in invertebrates is unknown. In the present study, an IL-16–like gene (EsIL-16) was identified from the Chinese mitten crab Eriocheir sinensis. EsIL-16 was predicted to encode a precursor (proEsIL-16) that shares similarities with pro-IL-16 proteins from insects and vertebrates. We show that caspase-3 processes proEsIL-16 into an approximately 144-kDa N-terminal prodomain with nuclear import activity and an approximately 34-kDa mature peptide that might be secreted into the extracellular region. EsIL-16 mRNA could be detected in all analyzed tissues and was significantly upregulated after immune challenge both in vitro and in vivo. T7 phage display library screening suggested potential binding activity between EsIL-16 and integrin, which was confirmed by coimmunoprecipitation assay. Interestingly, EsIL-16 promoted cell proliferation via integrin β1 in primary cultured crab hemocytes and Drosophila S2 cells. Furthermore, the interaction between EsIL-16 and integrin β1 was necessary to efficiently protect the host from bacterial infection. To our knowledge, this study revealed integrin β1 as a receptor for IL-16 and the function of this interaction in hemocyte proliferation in invertebrates for the first time. These results provide new insights into the regulation of innate immune responses in invertebrates and shed the light on the evolution of ILs within the animal kingdom.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

JAM-L–mediated leukocyte adhesion to endothelial cells is regulated in cis by α4β1 integrin activation

Junctional adhesion molecules (JAMs) are endothelial and epithelial adhesion molecules involved in the recruitment of circulating leukocytes to inflammatory sites. We show here that JAM-L, a protein related to the JAM family, is restricted to leukocytes and promotes their adhesion to endothelial cells. Cis dimerization of JAM-L is required to engage in heterophilic interactions with its cognate counter-receptor CAR (coxsackie and adenovirus receptor). Interestingly, JAM-L expressed on neutrophils binds CAR independently of integrin activation. However, on resting monocytes and T lymphocytes, which express the integrin VLA-4, JAM-L molecules engage in complexes with VLA-4 and mainly accumulate in their monomeric form. Integrin activation is required for the dissociation of JAM-L–VLA-4 complexes and the accumulation of functional JAM-L dimers, which indicates that the leukocyte integrin VLA-4 controls JAM-L function in cis by controlling its dimerization state. This provides a mechanism through which VLA-4 and JAM-L functions are coordinately regulated, allowing JAM-L to strengthen integrin-dependent adhesion of leukocytes to endothelial cells.     

Interplay between tau and α‐synuclein liquid–liquid phase separation

In Parkinson''s disease with dementia, up to 50% of patients develop a high number of tau‐containing neurofibrillary tangles. Tau‐based pathologies may thus act synergistically with the α‐synuclein pathology to confer a worse prognosis. A better understanding of the relationship between the two distinct pathologies is therefore required. Liquid–liquid phase separation (LLPS) of proteins has recently been shown to be important for protein aggregation involved in amyotrophic lateral sclerosis, whereas tau phase separation has been linked to Alzheimer''s disease. We therefore investigated the interaction of α‐synuclein with tau and its consequences on tau LLPS. We find α‐synuclein to have a low propensity for both, self‐coacervation and RNA‐mediated LLPS at pH 7.4. However, full‐length but not carboxy‐terminally truncated α‐synuclein efficiently partitions into tau/RNA droplets. We further demonstrate that Cdk2‐phosphorylation promotes the concentration of tau into RNA‐induced droplets, but at the same time decreases the amount of α‐synuclein inside the droplets. NMR spectroscopy reveals that the interaction of the carboxy‐terminal domain of α‐synuclein with the proline‐rich region P2 of tau is required for the recruitment of α‐synuclein into tau droplets. The combined data suggest that the concentration of α‐synuclein into tau‐associated condensates can contribute to synergistic aSyn/tau pathologies.     

A short C-terminal peptide in Gγ regulates Gβγ signaling efficacy

G protein beta-gamma (Gβγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM localization and functioning of Gβγ, allowing GPCR-G protein signaling to proceed. The diverse Gγ family has 12 members, and we have recently shown that the signaling efficacies of major Gβγ effectors are Gγ-type dependent. This dependency is due to the distinct series of membrane-interacting abilities of Gγ. However, the molecular process allowing for Gβγ subunits to exhibit a discrete and diverse range of Gγ-type–dependent membrane affinities is unclear and cannot be explained using only the type of prenylation. The present work explores the unique designs of membrane-interacting CT residues in Gγ as a major source for this Gγ-type–dependent Gβγ signaling. Despite the type of prenylation, the results show signaling efficacy at the PM, and associated cell behaviors of Gβγ are governed by crucially located specific amino acids in the five to six residue preprenylation region of Gγ. The provided molecular picture of Gγ–membrane interactions may explain how cells gain Gγ-type–dependent G protein-GPCR signaling as well as how Gβγ elicits selective signaling at various subcellular compartments.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

A model for how Gβγ couples Gα to GPCR

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine–proline–phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR–G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ–rhodopsin interactions may facilitate GPCR dimer transactivation.     

Lytic Effects of Staphylococcal α-Toxin and δ-Hemolysin

Several preparations of staphylococcal alpha-toxin and delta-lysin were studied in order to compare hemolytic activity with capacity to lyse bacterial protoplasts. delta-Lysin in relatively low concentration lysed protoplasts of Sarcina lutea, protoplasts of Streptococcus faecalis, and spheroplasts of Escherichia coli. Lysis of bacterial protoplasts by preparations of alpha-toxin appeared to be due to contamination of the preparations with delta-lysin. Data comparing the protoplast-lysing activity of various lytic agents are presented.     

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη''s function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη''s TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη''s function.     

The Laminin α Chains: Expression, Developmental Transitions, and Chromosomal Locations of α1-5, Identification of Heterotrimeric Laminins 8–11, and Cloning of a Novel α3 Isoform

Laminin trimers composed of α, β, and γ chains are major components of basal laminae (BLs) throughout the body. To date, three α chains (α1–3) have been shown to assemble into at least seven heterotrimers (called laminins 1–7). Genes encoding two additional α chains (α4 and α5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant α4 and α5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that α4 and α5 assemble into four novel laminin heterotrimers (laminins 8–11: α4β1γ1, α4β2γ1, α5β1γ1, and α5β2γ1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of α1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, α4 and α5 exhibited the broadest patterns of expression, while expression of α1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the α chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one α chain, all α chains were present in multiple BLs, and some BLs contained two or three α chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different α chains and two different β chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five α chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length α3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.