Mitochondria–lysosome interactions are essential for maintaining intracellular homeostasis. Although various fluorescent probes have been developed to visualize such interactions, they remain unable to label mitochondria and lysosomes simultaneously and dynamically track their interaction. Here, we introduce a cell-permeable, biocompatible, viscosity-responsive, small organic molecular probe, Coupa, to monitor the interaction of mitochondria and lysosomes in living cells. Through a functional fluorescence conversion, Coupa can simultaneously label mitochondria with blue fluorescence and lysosomes with red fluorescence, and the correlation between the red–blue fluorescence intensity indicates the progress of mitochondria–lysosome interplay during mitophagy. Moreover, because its fluorescence is sensitive to viscosity, Coupa allowed us to precisely localize sites of mitochondria–lysosome contact and reveal increases in local viscosity on mitochondria associated with mitochondria–lysosome contact. Thus, our probe represents an attractive tool for the localization and dynamic tracking of functional mitochondria–lysosome interactions in living cells.

Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics targets damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.

In a transparent medium, lensbased optical microscopy could focus a coherent light beam (e.g., laser) into a tiny spot, whose dimension is comparable to the size of the wavelength of the light. The diameter of the smallest beam waist is about half the size of the wavelength, which is referred to as the diffraction limit. Thus, for visible light, the theoretical diffraction limit is between 200 and 400 nm, much smaller than the size of a single cell (Figure 1A). However, in biological tissues that significantly scatter and absorb visible light, the spatial resolution could be compromised. In multicellular organisms, light absorption limits the penetration depth. The scattering of the light by the opaque biological tissues would expand the volume of light stimulation and reduce the spatial resolution.

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15Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple 16 signaling circuits. Channelrhodopsin-based optogenetics shows feasibility of stimulating neural 17 repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, 18 optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to 19 neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not 20 only enhanced axon regeneration in both regeneration competent and incompetent sensory 21 neurons in the peripheral nervous system, but also allowed temporal tuning and proper guidance 22 of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during 23 2 regeneration, showing a gated versus graded response, respectively. Importantly in the central 24 nervous system, their activation promotes axon regrowth and functional recovery of the 25 thermonociceptive behavior. We conclude that non-neuronal optogenetics target damaged 26 neurons and signaling subcircuits, providing a novel strategy in the intervention of neural 27 damage with improved precision. 28 29 92 5 the previous optogenetic AKT system (Ong et al. 2016) with two tandom CIBNs (referred to as 93 optoAKT in this work) (Supplemental Fig. S1A). Consistent with previous studies, the association 94 of CIBN and CRY2 took about 1 second and the CIBN-CRY2 complex dissociated in the dark 95 within 10 minutes (Kennedy et al. 2010; Zhang et al. 2014). The fusion of Raf or AKT does not 96 affect the association and dissociation kinetics of CIBN and CRY2 and multiple cycles of CRY2-97 CIBN association and dissociation can be triggered by alternating light-dark treatment 98 (Supplemental Fig. S1B-S1D, Movie S1, S3). Activation of optoRaf and optoAKT resulted in 99 nuclear translocation of ERK-EGFP (Fig. 1A, Movie S2) and nuclear export of FOXO3-EGFP 100 (Fig. 1B, Movie S4) resolved by live-cell fluorescence imaging, indicative of activation of the 101 ERK and AKT signaling pathways, respectively.102Western blot analysis on pERK (activated by optoRaf) in HEK293 cells showed that 103 pERK activity (Fig. 1C) increased within 10 min blue light stimulation and returned to the basal 104 level 30 min after the blue light was shut off (Fig. 1D). There was a slight decrease of pERK 105 activity upon optoRaf activation for over 10 min, likely due to a negative feedback, which has 106 been consistently observed in previous studies (Zhou et al. 2017). On the other hand, continuous 107 light illumination maintained a sustained activation of pCRY2-mCh-AKT (referred to as 108 optoAKT in this work) within an onset of 10 min (Fig. 1E). The inactivation kinetics of pAKT 109 was 30 min, similar to that of pERK ( Fig. 1F, 1G). Note we use only the phosphorylated and 110 total forms of CRY2-mCh-AKT to quantify the light response of optoAKT because the 111 endogenous AKT does not respond to light.112 113 optoRaf and optoAKT do not show crosstalk activity at the pERK and pAKT level 114 ...

The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP2 lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.

Summary The past decade has witnessed enormous progress in optogenetics, which uses photo‐sensitive proteins to control signal transduction in live cells and animals. The ever‐increasing amount of optogenetic tools, however, could overwhelm the selection of appropriate optogenetic strategies. In this work, we summarize recent progress in this emerging field and highlight the application of opsin‐free optogenetics in studying embryonic development, focusing on new insights gained into optical induction of morphogenesis, cell polarity, cell fate determination, tissue differentiation, neuronal regeneration, synaptic plasticity, and removal of cells during development.