Side-chain-engineered fluorescent dyes for 3D and long-term dynamic tracking of the plasma membrane in living cells.

The plasma membrane involves in many important biological events such as cell fusion and programmed cell death, but most of current plasma membrane probes cannot meet the requirement of long-term specific anchoring to the plasma membrane. Herein, we propose a molecular side-chain engineering strategy to modulate the long-term imaging performance of fluorescent dyes to the plasma membrane by regulating the cell permeability and anchoring ability. A series of FMR dyes with different lengths of side chains were designed and synthesized, and their transmembrane behaviours and staining performance were evaluated in living HeLa cells. We found that short-chain and medium-chain FMR dyes have excellent cell permeability without the labeling ability to the plasma membrane while the long-chain FMR dyes specifically stain the plasma membrane and can be firmly anchored to the plasma membrane for a long period of time. These long-chain FMR dyes have high stain specificality to the plasma membrane, and C10-FMR can be anchored to the plasma membrane of living cells for 2 h, which enables it to continuously monitor dynamic changes of the plasma membrane. The three-dimensional precision imaging of various cells was achieved using C10-FMR, which provides an opportunity to obtain complete information on the three-dimensional spatial morphology of the plasma membrane. The PEG-induced cell fusion of chicken red blood cells and H2O2-induced apoptosis of HeLa cells were monitored by real-time tracking of dynamic changes of the plasma membrane during these processes, which provide solid examples to prove the usefulness of these fluorescent dyes as long-term imaging tools. This work validates the hypothesis that cell permeability of membrane dyes can be readily regulated by tuning the side chains, and provides the effective design strategy of fluorescent dyes for 3D and long-term dynamic tracking of the plasma membrane of diverse animal cells.

Charge-regulated fluorescent anchors enable high-fidelity tracking of plasma membrane dynamics during biological events.

Many biological processes generally require long-term visualization tools for time-scale dynamic changes of the plasma membrane, but there is still a lack of design rules for such imaging tools based on small-molecule fluorescent probes. Herein, we revealed the key regulatory roles of charge number and species of fluorescent dyes in the anchoring ability of the plasma membrane and found that the introduction of multi-charged units and appropriate charge species is often required for fluorescent dyes with strong plasma membrane anchoring ability by systematically investigating the structure-function relationship of cyanostyrylpyridium (CSP) dyes with different charge numbers and species and their imaging performance for the plasma membrane. The CSP-DBO dye constructed exhibits strong plasma membrane anchoring ability in staining the plasma membrane of cells, in addition to many other advantages such as excellent biocompatibility and general universality of cell types. Such a fluorescent anchor has been successfully used to monitor chemically induced plasma membrane damage and dynamically track various cellular biological events such as cell fusion and cytokinesis over a long period of time by continuously monitoring the dynamic morphological changes of the plasma membrane, providing a valuable precise visualization tool to study the physiological response to chemical stimuli and reveal the structural morphological changes and functions of the plasma membrane during these important biological events from a dynamic perspective. Furthermore, CSP-DBO exhibits excellent biocompatibility and imaging capability in vivo such as labelling the plasma membrane in vivo and monitoring the metabolic process of lipofuscin as an aging indicator.

From phenotype to mechanism: Prenatal spectrum of NKAP mutation-related disorder and its pathogenesis inducing congenital heart disease.

NKAP mutations are associated with Hackmann-Di Donato-type X-linked syndromic intellectual developmental disorder (MRXSHD, MIM: #301039). Here, we elucidate the potential prenatal manifestation of NKAP mutation-associated disorder for the first time, alongside revealing the relationship between NKAP mutations and congenital heart defect (CHD) in the Chinese population. An NKAP mutation (NM_024528.4: c.988C>T, p.Arg330Cys) was identified in two foetuses presenting with CHD. Subsequent mechanistic exploration revealed a marked downregulation of NKAP transcription within HEK293T cells transfected with NKAP p.R330C. However, no significant change was observed at the protein level. Moreover, the mutation led to a dysregulation in the transcription of genes associated with cardiac morphogenesis, such as DHRS3, DNAH11 and JAG1. Additionally, our research determined that NKAP p.R330C affected Nkap protein intra-nuclear distribution, and binding with Hdac3. Summarily, our study strengthens NKAP mutations as a cause of CHD and prompts the reclassification of NKAP p.R330C as likely pathogenic, thereby establishing a prospective prenatal phenotypic spectrum that provides new insight into the prenatal diagnosis of CHD. Our findings also provide evidence of NKAP p.R330C pathogenicity and demonstrate the potential mechanism by which p.R330C dysregulates cardiac developmental gene transcription by altering Nkap intra-nuclear distribution and obstructing the interaction between Nkap and Hdac3, thereby leading to CHD.