Physical stimuli can act in either a synergistic or antagonistic manner to regulate cell fate decisions, but it is less clear whether insoluble signals alone can direct human pluripotent stem (hPS) ...cell differentiation into specialized cell types. We previously reported that stiff materials promote nuclear localization of the Yes-associated protein (YAP) transcriptional coactivator and support long-term self-renewal of hPS cells. Here, we show that even in the presence of soluble pluripotency factors, compliant substrata inhibit the nuclear localization of YAP and promote highly efficient differentiation of hPS cells into postmitotic neurons. In the absence of neurogenic factors, the effective substrata produce neurons rapidly (2 wk) and more efficiently (> 75%) than conventional differentiation methods. The neurons derived from substrate induction express mature markers and possess action potentials. The hPS differentiation observed on compliant surfaces could be recapitulated on stiff surfaces by adding small-molecule inhibitors of F-actin polymerization or by depleting YAP. These studies reveal that the matrix alone can mediate differentiation of hPS cells into a mature cell type, independent of soluble inductive factors. That mechanical cues can override soluble signals suggests that their contributions to early tissue development and lineage commitment are profound.
Deciphering the structural requirements and mechanisms for internalization of cell‐penetrating peptides (CPPs) is required to improve their delivery efficiency. Herein, a unique role of tryptophan ...(Trp) residues in the interaction and structuring of cationic CPP sequences with glycosaminoglycans (GAGs) has been characterized, in relation with cell internalization. Using isothermal titration calorimetry, circular dichroism, NMR, mass spectrometry, and phase‐contrast microscopy, we compared the interaction of 7 basic CPPs with 5 classes of GAGs. We found that the affinity of CPPs for GAGs increases linearly with the number of Trp residues, from 30 nM for a penetratin analog with 1 Trp residue to 1.5 nM for a penetratin analog with 6 Trp residues for heparin (HI); peptides with Trp residues adopt a predominantly β‐strand structure in complex with HI and form large, stable β‐sheet aggregates with GAGs; and in the absence of any cytotoxicity effect, the quantity of peptide internalized into CHO cells increased 2 times with 1 Trp residue, 10 times with 2 Trp residues, and 20 times with 3 Trp residues, compared with +6 peptides with no Trp residues. Therefore, Trp residues represent molecular determinants in basic peptide sequences not only for direct membrane translocation but also for efficient endocytosis through GAGs.—Bechara, C., Pallerla, M., Zaltsman, Y., Burlina, F., Alves, I. D., Lequin, O., Sagan S. Tryptophan within basic peptide sequences triggers glycosaminoglycan‐dependent endocytosis. FASEB J. 27, 738–749 (2013). www.fasebj.org
Leveraging the extraordinary potential of human pluripotent stem cells (hPSCs) requires an understanding of the mechanisms underlying cell-fate decisions. Substrate elasticity can induce ...differentiation by signaling through the transcriptional coactivator Yes-associated protein (YAP). Cells cultured on surfaces mimicking brain elasticity exclude YAP from their nuclei and differentiate to neurons. How YAP localization is controlled during neural differentiation has been unclear. We employed CRISPR/Cas9 to tag endogenous YAP in hPSCs and used this fusion protein to identify YAP's interaction partners. This engineered cell line revealed that neural differentiation promotes a change in YAP interactors, including a dramatic increase in angiomotin (AMOT) interaction with YAP. AMOT regulates YAP localization during differentiation. AMOT expression increases during neural differentiation and leads to YAP nuclear exclusion. Our findings that AMOT-dependent regulation of YAP helps direct hPSC fate provide insight into the molecular mechanisms by which the microenvironment can induce neural differentiation.
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•Endogenous tagging reveals YAP interactors in hPSCs•AMOT-YAP complex concentration increases during neural differentiation•AMOT regulates YAP localization in hPSCs•hPSC cytoskeleton influences YAP localization via AMOT
Kiessling and colleagues employed CRISPR/Cas9 to generate an hPSC line in which YAP is tagged, thus facilitating affinity purification of endogenous-level YAP complexes for analysis. This strategy uncovered proteins that interact with YAP during self-renewal and differentiation and identified angiomotin as a key regulator of YAP localization during neural differentiation.
Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this ...primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.
Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG(neg)) and sialic acid-deficient (SA(neg)) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.
Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA(neg)>WT>GAG(neg). The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA(neg) and GAG(neg) cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG(neg) cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.
These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses ...rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.
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•In vivo analysis of autism genes reveals a convergent vulnerability in neurogenesis•Systems biological analysis implicates the inner cortical plate and subventricular zone•Estrogen can mitigate the effects of disparate autism gene mutations•Estrogen inhibits Sonic hedgehog signaling
Using parallel in vivo analyses and systems biological approaches, Willsey et al. implicate cortical neurogenesis as a point of convergent vulnerability in autism spectrum disorders. They identify estrogen as a resilience factor for multiple, disparate autism genes and reveal a conserved role for estrogen in repressing Sonic hedgehog signaling.
Gene ontology analyses of high-confidence autism spectrum disorder (ASD) risk genes highlight chromatin regulation and synaptic function as major contributors to pathobiology. Our recent functional ...work in vivo has additionally implicated tubulin biology and cellular proliferation. As many chromatin regulators, including the ASD risk genes ADNP and CHD3, are known to directly regulate both tubulins and histones, we studied the five chromatin regulators most strongly associated with ASD (ADNP, CHD8, CHD2, POGZ and KMT5B) specifically with respect to tubulin biology. We observe that all five localize to microtubules of the mitotic spindle in vitro in human cells and in vivo in Xenopus. Investigation of CHD2 provides evidence that mutations present in individuals with ASD cause a range of microtubule-related phenotypes, including disrupted localization of the protein at mitotic spindles, cell cycle stalling, DNA damage and cell death. Lastly, we observe that ASD genetic risk is significantly enriched among tubulin-associated proteins, suggesting broader relevance. Together, these results provide additional evidence that the role of tubulin biology and cellular proliferation in ASD warrants further investigation and highlight the pitfalls of relying solely on annotated gene functions in the search for pathological mechanisms.
Human pluripotent stem (hPS) cells possess the capacity to self-renew indefinitely and differentiate into virtually all cell types. hPS cells thus represent an unlimited source of cells with ...potentially transformative applications such as cell-based regenerative medicine and drug discovery. These applications, however, require robust methods for directing the differentiation of hPS cells to desired cell types. To exert control over hPS cell fate, it is imperative to understand the signal inputs that hPS cells receive from their microenvironment. While initially the focus was on elucidating the role of soluble factors such as growth factors and small molecules, insoluble cues emanating from interactions between cells as well as between cells and the extracellular matrix are also important in contributing to hPS cell fate decisions. We found that, even in the presence of soluble factors that promote pluripotency, compliant substrates—with elasticity similar to human brain tissue—override these signals to induce efficient differentiation of hPS cells to neurons. Culture on soft substrates also augments soluble-factor based differentiation of hPS cells to motor neurons. The underlying molecular mechanism relies on F-actin and the transcriptional coactivator Yes-associated protein (YAP). Inhibition of F-actin polymerization or YAP depletion in hPS cells on a stiff substrate phenocopies the neuronal differentiation on a soft substrate. Our findings indicate that by modulating YAP localization, matrix elasticity can profoundly influence hPS cell fate. Next, I identified YAP interactors in hPS cells and characterized which interactions change during differentiation. I found that during neuronal differentiation YAP localization is regulated by angiomotin (AMOT). AMOT-YAP complexes increase during differentiation and forced expression of AMOT excludes YAP from the nucleus. These results suggest an overlooked role of AMOT in neurogenesis. Finally, I identified novel YAP interactors and generated hPS cell lines that facilitate monitoring of YAP interactions and localization in live cells. Cumulatively, my findings elucidate YAP’s role in hPS cell mechanosensing and cell fate control.
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