Plasma membrane (PM) stains are important organelle markers for monitoring membrane morphology and dynamics. The state-of-the-art PM stains are bright, specific, fluorogenic, and compatible with superresolution imaging. However, when recording membrane dynamics using advanced fluorescence microscopes, PM is prone to photodynamic damage introduced by dyes due to its phospholipid bilayer nature. Here, we introduce PK Mem dyes tailored for time-lapse fluorescence imaging. By integrating triplet-state quenchers into the MemBright dyes featuring cyanine chromophores and amphiphilic zwitterion anchors, PK Mem dyes exhibited a three-fold reduction in phototoxicity and a more than four-fold improvement in photostability in imaging experiments compared to MemBright prototypes. These dyes enable 2D and 3D imaging of live or fixed cancer cell lines and a wide range of primary cells, at the same time pair well with various fluorescent markers. PK Mem dyes can be applied to neuronal imaging in brain slices and in vivo two-photon imaging. The gentle nature of PK Mem palette enables ultralong-term recording of cell migration, cardiomyocyte beating, spermiogenesis, and axonal growth cone dynamics, which are prohibitively challenging using traditional PM dyes. Notably, PK Mem dyes are optically compatible with STED/SIM imaging, which can handily upgrade the routine of time-lapse neuronal imaging, such as growth cone tracking and mitochondrial transportations, into nanoscopic resolutions.

Sex determination in animals is not only determined by karyotype but can also be modulated by environmental cues like temperature via unclear transduction mechanisms. Moreover, in contrast to earlier views that sex may exclusively be determined by either karyotype or temperature, recent observations suggest that these factors rather co-regulate sex, posing another mechanistic mystery. Here, we discovered that certain wild-isolated and mutant C. elegans strains displayed genotypic germline sex determination (GGSD), but with a temperature-override mechanism. Further, we found that BiP, an ER chaperone, transduces temperature information into a germline sex-governing signal, thereby enabling the coexistence of GGSD and temperature-dependent germline sex determination (TGSD). At the molecular level, increased ER protein-folding requirements upon increased temperatures lead to BiP sequestration, resulting in ERAD-dependent degradation of the oocyte fate-driving factor, TRA-2, thus promoting male germline fate. Remarkably, experimentally manipulating BiP or TRA-2 expression allows to switch between GGSD and TGSD. Physiologically, TGSD allows C. elegans hermaphrodites to maintain brood size at warmer temperatures. Moreover, BiP can also influence germline sex determination in a different, non-hermaphroditic nematode species. Collectively, our findings identify thermosensitive BiP as a conserved temperature sensor in TGSD, and provide mechanistic insights into the transition between GGSD and TGSD.

Myristoylation is a type of lipidation with important functions. Owing to the lack of high-quality antibodies against myristoylation, developing alternative methods for profiling myristoylated proteins is important. Here, we provide a protocol for metabolic labeling using click chemistry to profile myristoylated proteins in C. elegans. Our approach improves the signal/noise ratio by covalently linking the myristoylated proteins to the beads. This protocol provides a highly specific and reproducible way for enriching myristoylated proteins, which could be modified to analyze other types of lipidations.

The brain monitors changes in the gut microbiome to maintain health, but the impact of specific bacterial alterations, as well as the underlying mechanisms, remains largely unclear. Here, we discovered an unexpected neuronal regulation of iron metabolism, mediating the neuronal surveillance of gut bacterial activity in C. elegans . Specifically, through a genome-wide screen, we identified 29 E. coli genes, mainly linked to metabolism regulation, whose inactivation could increase dopamine and serotonin biosynthesis in C. elegans head neurons. These neurons respond to the lack of respiratory chain genes in E. coli in the gut by perceiving intestinal mitochondrial impairment resulting from bacterial-induced reduction in host labile iron levels. Such neuronal responses subsequently promote intestinal ferritin-1 expression to counteract bacterially induced labile iron reduction, thus maintaining mitochondrial function. Our findings reveal how alterations in bacterial metabolism can elevate dopamine and serotonin levels in the host brain, demonstrating that the nervous system not only senses microbiome-caused changes in the gut but also feeds back to revert them.