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Hemithioindigos for cellular photopharmacology: desymmetrised molecular switch scaffolds enabling design control over the isomer-dependency of potent antimitotic bioactivity
摘要： Druglike small molecules with photoswitchable bioactivity – photopharmaceuticals – allow biologists to perform studies with exquisitely precise and reversible, spatial and temporal control over critical biological systems inaccessible to genetic manipulation. The photoresponsive pharmacophores disclosed have been almost exclusively azobenzenes, which has limited the structural and substituent scope of photopharmacology. More detrimentally, for azobenzene reagents, it is not researchers’ needs for adapted experimental tools, but rather protein binding site sterics, that typically force whether the trans (dark) or cis (lit) isomer is the more bioactive. We now present first hemithioindigo-based pharmacophores enabling photoswitchable control over endogenous biological activity in cellulo. HOTubs optically control microtubule depolymerisation and cell death in unmodified mammalian cells. Notably, we show how the asymmetry of hemithioindigos allows a priori design of either Z- or E- (dark- or lit)-toxic antimitotics, whereas the corresponding azobenzenes are exclusively lit-toxic. We thus demonstrate that hemithioindigos enable an important expansion of the substituent and design scope of photopharmacological interventions for biological systems.
关键词： Photopharmacology,Drug design,Photochromism,Cytoskeleton,Antiproliferation
更新于2025-09-23 15:23:52
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A genetically encoded FRET biosensor for visualizing EphA4 activity in different compartments of the plasma membrane
摘要： The EphA4 receptor tyrosine kinase is well known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensor based on F?rster resonance energy transfer (FRET), and targeted it in or outside raft-like microdomains in the plasma membrane. We showed that our biosensor can produce a robust and specific FRET response upon EphA4 activation, both in vitro and in live cells. Interestingly, we observed stronger FRET responses for the non-raft targeting biosensor than for the raft targeting biosensor, suggesting that stronger EphA4 activation may occur in non-raft regions. Further investigations revealed the importance of the actin cytoskeleton in suppressing EphA4 activity in raft-like microdomains. Therefore, our FRET-based EphA4 biosensor could serve as a powerful tool to visualize and investigate EphA4 activation and signaling in specific subcellular compartments of single live cells.
关键词： EphA4,Membrane microdomain,cytoskeleton,FRET biosensor,Live cell imaging
更新于2025-09-23 15:22:29
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Dynamic nanoscale morphology of the ER surveyed by STED microscopy
摘要： The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.
关键词： STED microscopy,Climp63,nanoholes,endoplasmic reticulum,reticulon,microtubule cytoskeleton
更新于2025-09-10 09:29:36
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Photoreceptor actin dysregulation in syndromic and non-syndromic retinitis pigmentosa
摘要： Retinitis pigmentosa (RP) is the leading cause of inherited blindness. RP is a genetically heterogeneous disorder, with more than 100 different causal genes identified in patients. Central to disease pathogenesis is the progressive loss of retinal photoreceptors. Photoreceptors are specialised sensory neurons that exhibit a complex and highly dynamic morphology. The highly polarised and elaborated architecture of photoreceptors requires precise regulation of numerous cytoskeletal elements. In recent years, significant work has been placed on investigating the role of microtubules (specifically, the acetylated microtubular axoneme of the photoreceptor connecting cilium) and their role in normal photoreceptor function. This has been driven by the emerging field of ciliopathies, human diseases arising from mutations in genes required for cilia formation or function, of which RP is a frequently reported phenotype. Recent studies have highlighted an intimate relationship between cilia and the actin cytoskeleton. This review will focus on the role of actin in photoreceptors, examining the connection between actin dysregulation in RP.
关键词： Espin,RP2,Myosin VIIa,photoreceptor,CRB1,cytoskeleton,actin,Retinitis pigmentosa,Whirlin,FSCN2,cilia,RPGR,PROM1
更新于2025-09-09 09:28:46