Exploiting Endogenous Enzymes for Cancer-Cell Selective Metabolic Labeling of RNA in Vivo.

Tissues and organs are composed of many diverse cell types, making cell-specific gene expression profiling a major challenge. Herein we report that endogenous enzymes, unique to a cell of interest, can be utilized to enable cell-specific metabolic labeling of RNA. We demonstrate that appropriately designed "caged" nucleosides can be rendered active by serving as a substrate for cancer-cell specific enzymes to enable RNA metabolic labeling, only in cancer cells. We envision that the ease and high stringency of our approach will enable expression analysis of tumor cells in complex environments.

Introducing immunohistochemistry to the molecular biology laboratory.

Widely used in research laboratories, immunohistochemistry (IHC) is a transferable skill that prepares undergraduate students for a variety of careers in the biomedical field. We have developed an inquiry-based learning IHC laboratory exercise, which introduces students to the theory, procedure, and data interpretation of antibody staining. Students are tasked with performing IHC using an "unknown" antibody and then asked to identify the cells or molecular structures within the nervous systems specific for that unknown antibody. In two lab sessions, students are exposed to handling of delicate brain slices, fluorescent microscopy, and data analysis using the Allen Brain Atlas (ABA), an online freely accessible database of mRNA transcript expression patterns in the brain. Here, we present guidelines for easy implementation in the classroom and assess learning gains achieved by the students upon completion of the IHC laboratory module. Students clearly displayed an increase in knowledge in data interpretation, procedural knowledge, and theory surrounding IHC. Thus, this module works as an inquiry-based learning based method to introduce IHC principles to undergraduate students.

Single cell transcriptomics of human epidermis identifies basal stem cell transition states.

How stem cells give rise to epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find four spatially distinct stem cell populations at the top and bottom of rete ridges and transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling suggests that basal cell populations serve as crucial signaling hubs to maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest that transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.

Defining the Genetics of Basosquamous Carcinoma.

Basosquamous carcinoma (BSC) is a rare form of skin cancer with both basaloid and squamous morphology. Chiang et al. (2019) genetically define BSCs and demonstrate that BSCs likely originate as basal cell carcinomas that partially squamatize through accumulation of ARID1A mutations and RAS/MAPK pathway activation.

MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias.

The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.

Actin polymerization controls cilia-mediated signaling.

Primary cilia are polarized organelles that allow detection of extracellular signals such as Hedgehog (Hh). How the cytoskeleton supporting the cilium generates and maintains a structure that finely tunes cellular response remains unclear. Here, we find that regulation of actin polymerization controls primary cilia and Hh signaling. Disrupting actin polymerization, or knockdown of N-WASp/Arp3, increases ciliation frequency, axoneme length, and Hh signaling. Cdc42, a potent actin regulator, recruits both atypical protein pinase C iota/lambda (aPKC) and Missing-in-Metastasis (MIM) to the basal body to maintain actin polymerization and restrict axoneme length. Transcriptome analysis implicates the Src pathway as a major aPKC effector. aPKC promotes whereas MIM antagonizes Src activity to maintain proper levels of primary cilia, actin polymerization, and Hh signaling. Hh pathway activation requires Smoothened-, Gli-, and Gli1-specific activation by aPKC. Surprisingly, longer axonemes can amplify Hh signaling, except when aPKC is disrupted, reinforcing the importance of the Cdc42-aPKC-Gli axis in actin-dependent regulation of primary cilia signaling.