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InDrosophila, two interacting adhesion protein families, Defective proboscis responses (Dprs) and Dpr interacting proteins (DIPs), coordinate the assembly of neural networks. While intercellular DIP::Dpr interactions have been well characterized, DIPs and Dprs are often co-expressed within the same cells, raising the question as to whether they also interact incis. We show, in cultured cells andin vivo, that DIP-α and DIP-δ can interact inciswith their ligands, Dpr6/10 and Dpr12, respectively. When co-expressed inciswith their cognate partners, these Dprs regulate the extent oftransbinding, presumably through competitivecisinteractions. We demonstrate the neurodevelopmental effects ofcisinhibition in fly motor neurons and in the mushroom body. We further show that a long disordered region of DIP-α at the C-terminus is required forcisbut nottransinteractions, likely because it alleviates geometric constraints oncisbinding. Thus, the balance betweencisandtransinteractions plays a role in controlling neural development. 

The cytoplasmic tails of classical cadherins form a multiprotein cadherin–catenin complex (CCC) that constitutes the major structural unit of adherens junctions (AJs). The CCC in AJs forms junctional clusters, “E clusters,” driven bycisandtransinteractions in the cadherin ectodomain and stabilized by α-catenin–actin interactions. Additional proteins are known to bind to the cytoplasmic region of the CCC. Here, we analyze how these CCC-associated proteins (CAPs) integrate into cadherin clusters and how they affect the clustering process. Using a cross-linking approach coupled with mass spectrometry, we found that the majority of CAPs, including the force-sensing protein vinculin, interact with CCCs outside of AJs. Accordingly, structural modeling shows that there is not enough space for CAPs the size of vinculin to integrate into E clusters. Using two CAPs, scribble and erbin, as examples, we provide evidence that these proteins form separate clusters, which we term “C clusters.” As proof of principle, we show, by using cadherin ectodomain monoclonal antibodies (mAbs), that mAb-bound E-cadherin forms separate clusters that undergotransinteractions. Taken together, our data suggest that, in addition to its role in cell–cell adhesion, CAP-driven CCC clustering serves to organize cytoplasmic proteins into distinct domains that may synchronize signaling networks of neighboring cells within tissues. 

Abstract Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition.DrosophilaDIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. We show here that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. We then describe a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, to reveal how specificity is coded on the canonical DIP/Dpr interface. We show that binding specificity of DIP/Dpr subgroups is controlled by “negative constraints”, which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. We discuss the structural origins of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families. 

We discovered that cells expressing the adherens junction protein nectin-1 capture nectin-4 containing membranes from the surface of adjacent cells in a trans-endocytosis process. Internalized nectin-1/4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1 expressing cells acquire dye-labelled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. 

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions.