A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice.

Advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2-g, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. The microscope had on-board photon detectors, robust to environmental light, and the arena lighting was timed to the end of each line-scan, enabling functional imaging of activity from cortical layer 4 and layer 6 neurons expressing jGCaMP7f in mice roaming a fully lit or dark arena.

A single PXXP motif in the C-terminal region of srGAP3 mediates binding to multiple SH3 domains.

The Slit-Robo GTPase-activating protein 3 (srGAP3) has been implicated in different critical aspects of neuronal development. These findings have mainly been based on the characterisation of the three conserved globular N-terminal domains, while the function of the C-terminal region (CTR) is still unknown. We show that this predicted unstructured region acts as an adaptor by binding to the endocytic proteins Amphiphysin, Endophilin-A2, Endophilin-A1, as well as the Ras signalling protein Grb2.

The role of conserved PEX3 regions in PEX19-binding and peroxisome biogenesis.

The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane.

Autoinhibition of GEF activity in Intersectin 1 is mediated by the short SH3-DH domain linker.

Intersectin 1L (ITSN1L) acts as a specific guanine nucleotide exchange factor (GEF) for the small guanine nucleotide binding protein Cdc42 via its C-terminal DH domain. Interestingly, constructs of ITSN1L that comprise additional domains, for instance the five SH3 domains amino-terminal of the DH domain, were shown to be inhibited in their exchange factor activity. Here, we investigate the inhibitory mechanism of ITSN1L in detail and identify a novel short amino acid motif which mediates autoinhibition.

1H, 13C, and 15N chemical shift assignments for the Eps15-EH2-stonin 2 complex.

EH domains are protein-protein interaction domains that function in vesicular trafficking and endocytosis. Here, we report the NMR spectral assignments of the high-affinity complex between the second EH domain of Eps15 and a stonin 2 peptide--providing the basis for the characterization of a two-site binding mode.

Isoform-selective interaction of the adaptor protein Tks5/FISH with Sos1 and dynamins.

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells.

Characterisation of the nucleotide exchange factor ITSN1L: evidence for a kinetic discrimination of GEF-stimulated nucleotide release from Cdc42.

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5'-diphosphate (GDP) to allow guanosine-5'-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42.

Spectroscopic methods for the determination of protein interactions.

This unit provides guidelines on how to use steady-state fluorescence spectroscopy for the quantification of protein-protein interactions. The fluorescence of a protein is characterized by its excitation and emission spectra, quantum yield, and anisotropy. These parameters can change upon interaction with another protein and can be used to measure the extent of complex formation.

Structure of the Eps15-stonin2 complex provides a molecular explanation for EH-domain ligand specificity.

Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity.

Cbl promotes clustering of endocytic adaptor proteins.

The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and beta-PIX in complex with a proline-arginine peptide from Cbl-b.

Balance of ATPase stimulation and nucleotide exchange is not required for efficient refolding activity of the DnaK chaperone.

The DnaK system from Thermus thermophilus (DnaK(Tth)) exhibits pronounced differences in organisation and regulation to its mesophile counterpart from Escherichia coli (DnaK(Eco)). While the ATPase cycle of DnaK(Eco) is tightly regulated by the concerted action of the two cofactors DnaJ(Eco) and GrpE(Eco), the DnaK(Tth) system features an imbalance in this cochaperone mediated regulation. GrpE(Tth) considerably accelerates the ATP/ADP exchange, but DnaJ(Tth) only slightly stimulates ATPase activity, believed to be a key step for chaperone activity of DnaK(Eco).

Activation and assembly of the NADPH oxidase: a structural perspective.

The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. The activity of this multi-protein enzyme is dependent on the regulated assembly of the six enzyme subunits at the membrane where oxygen is reduced to superoxide anions. In the resting state, four of the enzyme subunits are maintained in the cytosol, either through auto-inhibitory interactions or through complex formation with accessory proteins that are not part of the active enzyme complex.

DafA cycles between the DnaK chaperone system and translational machinery.

DafA is encoded by the dnaK operon of Thermus thermophilus and mediates the formation of a highly stable complex between the chaperone DnaK and its co-chaperone DnaJ under normal growth conditions. DafA(Tth) contains 87 amino acid residues and is the only member of the DnaK(Tth) chaperone system for which no corresponding protein has yet been identified in other organisms and whose particular function has remained elusive. Here, we show directly that the DnaK(Tth)-DnaJ(Tth)-DafA(Tth) complex cannot represent the active chaperone species since DafA(Tth) inhibits renaturation of firefly luciferase by suppressing substrate association.

Molecular basis of phosphorylation-induced activation of the NADPH oxidase.

The multi-subunit NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of reactive oxygen species. Activation of the NADPH oxidase requires the targeting of a cytoplasmic p40-p47-p67(phox) complex to the membrane bound heterodimeric p22-gp91(phox) flavocytochrome. This interaction is prevented in the resting state due to an auto-inhibited conformation of p47(phox).

The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity.

ClpB from Thermus thermophilus belongs to the Clp/Hsp100 protein family and reactivates protein aggregates in cooperation with the DnaK chaperone system. The mechanism of protein reactivation and interaction with the DnaK system remains unclear. ClpB possesses two nucleotide binding domains, which are essential for function and show a complex allosteric behavior.

Architecture of the p40-p47-p67phox complex in the resting state of the NADPH oxidase. A central role for p67phox.

The phagocyte NADPH oxidase is a multiprotein enzyme whose subunits are partitioned between the cytosol and plasma membrane in resting cells. Upon exposure to appropriate stimuli multiple phosphorylation events in the cytosolic components take place, which induce rearrangements in a number of protein-protein interactions, ultimately leading to translocation of the cytoplasmic complex to the membrane. To understand the molecular mechanisms that underlie the assembly and activation process we have carried out a detailed study of the protein-protein interactions that occur in the p40-p47-p67(phox) complex of the resting oxidase.