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.
Abstract-The K ϩ channel mKv1.5 is thought to encode a 4-aminopyridine (4-AP)-sensitive component of the current I K,slow in the mouse heart. We used gene targeting to replace mKv1.5 with the 4-AP-insensitive channel rKv1.1 (SWAP mice) and directly test the role of Kv1.5 in the mouse ventricle. Kv1.5 RNA and protein were undetectable, rKv1.1 was expressed, and Kv2.1 protein was upregulated in homozygous SWAP hearts. The density of the K ϩ current I K,slow (depolarizations to ϩ40 mV, pA/pF) was similar in left ventricular myocytes isolated from SWAP homozygotes (17Ϯ1, nϭ27) and littermate controls (16Ϯ2, nϭ19). The densities and properties of I peak , I to,f , I to,s , and I ss were also unchanged. In homozygous SWAP myocytes, the 50-mol/L 4-AP-sensitive component of I K,slow was absent (nϭ6), the density of the 20-mmol/L tetraethylammonium-sensitive component of I K,slow was increased (9Ϯ1 versus 5Ϯ1, PϽ0.05), and no 100-to 200-nmol/L ␣-dendrotoxin-sensitive current was found (nϭ8). APD 90 in SWAP myocytes was similar to controls at baseline but did not prolong in response to 30 mol/L 4-AP. Similarly, QTc (ms) was not prolonged in anesthetized SWAP mice (64Ϯ2, homozygotes, nϭ9; 62Ϯ2, controls, nϭ9), and injection with 4-AP prolonged QTc only in controls (63Ϯ1, homozygotes; 72Ϯ2, controls; PϽ0.05). SWAP mice had no increase in arrhythmias during ambulatory telemetry monitoring. Thus, Kv1.5 encodes the 4-AP-sensitive component of I K,slow in the mouse ventricle and confers sensitivity to 4-AP-induced prolongation of APD and QTc. Compensatory upregulation of Kv2.1 may explain the phenotypic differences between SWAP mice and the previously described transgenic mice expressing a truncated dominant-negative Kv1.
The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor–related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.
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