Delineating the heterogeneity of matrix-directed differentiation toward soft and stiff tissue lineages via single-cell profiling.

Mesenchymal stromal/stem cells (MSCs) form a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers that direct differentiation toward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of nonconditioned, matrix-conditioned, and early differentiating cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We show that soft matrices support adipogenesis of multipotent cells and early endochondral ossification of nonadipogenic cells, whereas intramembranous ossification and preosteoblast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed differentiation and perform whole-genome screening of mechanoresponsive genes. Specifically, top-ranked tropomyosin-1 is highly sensitive to stiffness cues both at RNA and protein levels, and changes in TPM1 expression determine the differentiation toward soft versus stiff tissue lineage. Consistent with actin stress fiber stabilization, tropomyosin-1 overexpression maintains YAP1 nuclear localization, activates YAP1 target genes, and directs osteogenic differentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, suppressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adipogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.

CloneSeq: A highly sensitive analysis platform for the characterization of 3D-cultured single-cell-derived clones.

Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.

Interleukin-1α dependent survival of cardiac fibroblasts is associated with StAR/STARD1 expression and improved cardiac remodeling and function after myocardial infarction.

AIMS: One unaddressed aspect of healing after myocardial infarction (MI) is how non-myocyte cells that survived the ischemic injury, keep withstanding additional cellular damage by stress forms typically arising during the post-infarction inflammation. Here we aimed to determine if cell survival is conferred by expression of a mitochondrial protein novel to the cardiac proteome, known as steroidogenic acute regulatory protein, (StAR/STARD1). Further studies aimed to unravel the regulation and role of the non-steroidogenic cardiac StAR after MI. METHODS AND RESULTS: Following permanent ligation of the left anterior descending coronary artery in mouse heart, timeline western blot analyses showed that StAR expression corresponds to the inflammatory response to MI. Following the identification of StAR in mitochondria of cardiac fibroblasts in culture, confocal microscopy immunohistochemistry (IHC) identified StAR expression in left ventricular (LV) activated interstitial fibroblasts, adventitial fibroblasts and endothelial cells. Further work with the primary fibroblasts model revealed that interleukin-1α (IL-1α) signaling via NF-κB and p38 MAPK pathways efficiently upregulates the expression of the Star gene products. At the functional level, IL-1α primed fibroblasts were protected against apoptosis when exposed to cisplatin mimicry of in vivo apoptotic stress; yet, the protective impact of IL-1α was lost upon siRNA mediated StAR downregulation. At the physiological level, StAR expression was nullified during post-MI inflammation in a mouse model with global IL-1α deficiency, concomitantly resulting in a 4-fold elevation of apoptotic fibroblasts. Serial echocardiography and IHC studies of mice examined 24 days after MI revealed aggravation of LV dysfunction, LV dilatation, anterior wall thinning and adverse tissue remodeling when compared with loxP control hearts. CONCLUSIONS: This study calls attention to overlooked aspects of cellular responses evolved under the stress conditions associated with the default inflammatory response to MI. Our observations suggest that LV IL-1α is cardioprotective, and at least one mechanism of this action is mediated by induction of StAR expression in border zone fibroblasts, which renders them apoptosis resistant. This acquired survival feature also has long-term ramifications on the heart recovery by diminishing adverse remodeling and improving the heart function after MI.

Vimentin protects differentiating stem cells from stress.

Vimentin is one of the first cytoplasmic intermediate filaments to be expressed in mammalian cells during embryogenesis, but its role in cellular fitness has long been a mystery. Vimentin is acknowledged to play a role in cell stiffness, cell motility, and cytoplasmic organization, yet it is widely considered to be dispensable for cellular function and organismal development. Here, we show that Vimentin plays a role in cellular stress response in differentiating cells, by recruiting aggregates, stress granules, and RNA-binding proteins, directing their elimination and asymmetric partitioning. In the absence of Vimentin, pluripotent embryonic stem cells fail to differentiate properly, with a pronounced deficiency in neuronal differentiation. Our results uncover a novel function for Vimentin, with important implications for development, tissue homeostasis, and in particular, stress response.

Mitochondrial dysfunction in protein conformational disorders.

Protein aggregation is a hallmark of many neurodegenerative diseases. In Parkinson's disease protein misfolding of α-synuclein involves conformational changes in the protein structure that often results in self-association and aggregation leading to accumulation of α-synuclein in neuronal cells. The underlying mechanisms by which aggregations can lead to impaired cellular functions are often not understood. Meanwhile, there is growing evidence that links mitochondrial dysfunction to Parkinson's disease. As both mitochondria and protein aggregation of α-synuclein have been shown to play a major role in Parkinson's disease, it seems likely that a converging mechanism exists that links the two pathways.

Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion.

The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion) to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE's success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE's direct measurement and ease of use make it appealing for cell biologists.

Imaging stress.

Recent innovations in cell biology and imaging approaches are changing the way we study cellular stress, protein misfolding, and aggregation. Studies have begun to show that stress responses are even more variegated and dynamic than previously thought, encompassing nano-scale reorganization of cytosolic machinery that occurs almost instantaneously, much faster than transcriptional responses. Moreover, protein and mRNA quality control is often organized into highly dynamic macromolecular assemblies, or dynamic droplets, which could easily be mistaken for dysfunctional "aggregates," but which are, in fact, regulated functional compartments. The nano-scale architecture of stress-response ranges from diffraction-limited structures like stress granules, P-bodies, and stress foci to slightly larger quality control inclusions like juxta nuclear quality control compartment (JUNQ) and insoluble protein deposit compartment (IPOD), as well as others. Examining the biochemical and physical properties of these dynamic structures necessitates live cell imaging at high spatial and temporal resolution, and techniques to make quantitative measurements with respect to movement, localization, and mobility. Hence, it is important to note some of the most recent observations, while casting an eye towards new imaging approaches that offer the possibility of collecting entirely new kinds of data from living cells.