No abstract

Neural stem cells (NSCs) generate neurons and glial cells throughout embryonic and postnatal brain development. The role of S-palmitoylation (also referred to as S-acylation), a reversible posttranslational lipid modification of proteins, in regulating the fate and activity of NSCs remains largely unknown. We used an unbiased screening approach to identify proteins that are S-acylated in mouse NSCs and showed that bone morphogenic protein receptor 1a (BMPR1a), a core mediator of BMP signaling, is palmitoylated. Genetic manipulation of S-acylated sites affects the localization and trafficking of BMPR1a and leads to altered BMP signaling. Strikingly, defective palmitoylation of BMPR1a modulates NSC function within the mouse brain, resulting in enhanced oligodendrogenesis. Thus, we identified a mechanism regulating the behavior of NSCs and provided the framework to characterize dynamic posttranslational lipid modifications of proteins in the context of NSC biology.

Summary This protocol describes the isolation and culturing of primary neural stem cells (NSCs) from the adult mouse hippocampus, followed by the experimental approach for fluorescence loss in photobleaching assays, previously used to characterize the presence of an endoplasmic reticulum (ER) membrane diffusion barrier. The assay described here can be used to study live asymmetry in the ER membrane or other organelles that is established in dividing NSCs. For complete details on the use and execution of this protocol, please refer to Clay et al. (2014) ; bin Imtiaz et al. (2021) ; Lee et al. (2016) ; Luedeke et al. (2005) ; Moore et al. (2015) ; Shcheprova et al. (2008) .

Asymmetric segregation of cellular components regulates the fate and behavior of somatic stem cells. Similar to dividing budding yeast and precursor cells in C. elegans, it has been shown that mouse neural progenitors establish a diffusion barrier in the membrane of the endoplasmic reticulum (ER), which has been associated with asymmetric partitioning of damaged proteins and cellular age. However, the existence of an ER-diffusion barrier in human cells remains unknown. Here we used fluorescence loss in photobleaching (FLIP) imaging to show that human embryonic stem cell (hESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells establish an ER-diffusion barrier during cell division. The human ER-diffusion barrier is regulated via Lamin-dependent mechanisms and is associated with asymmetric segregation of mono- and polyubiquitinated, damaged proteins. Further, forebrain regionalized organoids derived from hESCs were used to show the establishment of an ER-membrane diffusion barrier in more naturalistic tissues mimicking early steps of human brain development. Thus, the data provided here show that human neural progenitors establish a diffusion barrier during cell division in the membrane of the ER, which may allow for asymmetric segregation of cellular components, contributing to the fate and behavior of human neural progenitor cells.