SummaryHuman pluripotent stem cells (hPSCs) maintain a highly fragmented mitochondrial network, but the mechanisms regulating this phenotype remain unknown. Here, we describe a non-cell death function of the anti-apoptotic protein, MCL-1, in regulating mitochondrial dynamics and promoting pluripotency of stem cells. MCL-1 is induced upon reprogramming, and its inhibition or knockdown induces dramatic changes to the mitochondrial network as well as loss of the key pluripotency transcription factors, NANOG and OCT4. Aside from localizing at the outer mitochondrial membrane like other BCL-2 family members, MCL-1 is unique in that it also resides at the mitochondrial matrix in pluripotent stem cells. Mechanistically, we find MCL-1 to interact with DRP-1 and OPA1, two GTPases responsible for remodeling the mitochondrial network. Depletion of MCL-1 compromised the levels and activity of these key regulators of mitochondrial dynamics. Our findings uncover an unexpected, non-apoptotic function for MCL-1 in the maintenance of mitochondrial structure and stemness.

We have previously reported that mouse embryonic stem cells (mESCs) do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mESCs and mESC-differentiated cells. We demonstrate that LPS, TNFα, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NFκB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mESCs. Similar results were obtained in human ESCs (hESCs). In addition to the inactive state of NFκB, the deficiency of inflammatory response in mESCs is also attributed to the lack of functional receptors for LPS and TNFα. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NFκB from its inactive to active state. However, a limited response was observed in mESC-differentiated fibroblasts only to TNFα and viral infection, but not to LPS. We conclude that the inflammatory response mechanism is not active in mESCs, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this paper demonstrate that ESCs are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology and ESC-based regenerative medicine.

The core transcriptional network regulating stem cell self-renewal and pluripotency remains an intense area of research. Increasing evidence indicates that modified regulation of basic cellular processes such as mitochondrial dynamics, apoptosis, and cell cycle are also essential for pluripotent stem cell identity and fate decisions. Here, we review evidence for Wnt regulation of pluripotency and self-renewal, and its connections to emerging features of pluripotent stem cells, including (1) increased mitochondrial fragmentation, (2) increased sensitivity to cell death, and (3) shortened cell cycle. We provide a general overview of the stem cell–specific mechanisms involved in the maintenance of these uncharacterized hallmarks of pluripotency and highlight potential links to the Wnt signaling pathway. Given the physiological importance of stem cells and their enormous potential for regenerative medicine, understanding fundamental mechanisms mediating the crosstalk between Wnt, organelle-dynamics, apoptosis, and cell cycle will be crucial to gain insight into the regulation of stemness.

House dust mites are common pests with an unusual evolutionary history, being descendants of a parasitic ancestor. Transition to parasitism is frequently accompanied by genome rearrangements, possibly to accommodate the genetic change needed to access new ecology. Transposable element (TE) activity is a source of genomic instability that can trigger large-scale genomic alterations. Eukaryotes have multiple transposon control mechanisms, one of which is RNA interference (RNAi). Investigation of the dust mite genome failed to identify a major RNAi pathway: the Piwi-associated RNA (piRNA) pathway, which has been replaced by a novel small-interfering RNA (siRNA)-like pathway. Co-opting of piRNA function by dust mite siRNAs is extensive, including establishment of TE control master loci that produce siRNAs. Interestingly, other members of the Acari have piRNAs indicating loss of this mechanism in dust mites is a recent event. Flux of RNAi-mediated control of TEs highlights the unusual arc of dust mite evolution. Author summaryInvestigation of small RNA populations in dust mites revealed absence of the piwi-associated RNA (piRNA) pathway. Apart from several nematode and platyhelminths lineages, piRNAs are an essential component of animal genome surveillance, actively targeting and silencing transposable elements. In dust mites, expansion of Dicer produced small-interfering RNA (siRNA) biology compensates for loss of piRNAs. The dramatic difference we find in dust mites is likely a consequence of their evolutionary history, which is marked by descent from a parasite to the current free-living form. Our study highlights a correlation between perturbation of transposon surveillance and shifts in ecology.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.