Plexins are cell surface receptors that bind to semaphorins and transduce signals that regulate neuronal development, immune responses, and other processes. Signaling through plexins has been proposed to rely on specific GTPase activating protein (GAP) activity for R-Ras and M-Ras. Activation of this GAP activity appears to require simultaneous binding of semaphorin to the plexin extracellular domain and of a RhoGTPase to the cytoplasmic region. However, GAP activity of plexins has eluded detection in several recent studies. We show that the purified cytoplasmic region of plexin uses a non-canonical catalytic mechanism to act as a GAP for Rap, but not for R-Ras or M-Ras. The RapGAP activity of plexins was normally autoinhibited and could be activated by induced dimerization. Our biochemical and crystallographic analyses demonstrate that binding of the RhoGTPases did not directly contribute to activation of plexin RapGAP activity. Semaphorin stimulated the RapGAP activity of full-length plexin in cells, which was required for plexin-mediated neuronal growth cone collapse. These findings together define a pathway for plexin signaling and provide new insights into the mechanism for semaphorin-induced activation of plexin.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.

Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling.DOI: http://dx.doi.org/10.7554/eLife.01279.001

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Transport of intracellular cargo generally requires coiled-coil adaptor proteins that connect cargo-bound receptors, usually GTPases, to dynein motor complexes. Wang et al. report that two Rab GTPases, CRACR2a and Rab45, contain coiled-coil domains and can directly act as dynein adaptors with CRACR2a–dynein participating in calcium-regulated endocytic trafficking.

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Infrared-to-visible upconversion emission intensities are investigated in Li+ and Er3+ codoped ZnO nanocrystals. Li+ ions doped in ZnO/Er3+ nanocrystals can greatly enhance the upconversion emission intensity of Er3+ ions. The extended X-ray absorption fine structure spectroscopy data show that both the Er−O bond length and coordination number of the Er−Er bond have been altered by introducing Li+ ions in the ZnO/Er3+ nanocrystals. The variation of Er−O bond length leads to the change in the local asymmetry around Er3+ ions. Meanwhile, the greater coordination number of the Er−Er bond causes stronger interaction between neighboring Er3+ ions and, hence, strengthens the reaction Er3+ (4F7/2) + Er3+ (4I11/2) → 2Er3+ (4F9/2). In this process, the 4F9/2 state is a metastable state that could be excited to the 2H9/2 state by absorbing one photon at high excitation power. Li+ ions also can reduce the OH groups in specimen, which is the other reason for enhancing the upconversion emission intensities.

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.