A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.

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.

A Non-apoptotic Function of MCL-1 in Promoting Pluripotency and Modulating Mitochondrial Dynamics in Stem Cells.

Human 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.

Wnt Signaling and Its Impact on Mitochondrial and Cell Cycle Dynamics in Pluripotent Stem Cells.

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.

Apical polarization and lumenogenesis: The apicosome sheds new light.

Establishment of apico-basal polarity is critical for the lumenal epiblast-like morphogenesis of human pluripotent stem cells (hPSCs). In this issue, Taniguchi et al. (2017. J Cell Biol. https://doi.org/10.1083.jcb201704085) describe a structure called the apicosome, generated in single hPSCs, that allows them to self-organize and form the lumenal epiblast-like stage.

Chronicle of a Neuronal Death Foretold: Preventing Aging by Keeping MGRN1 at the Nucleus.

In this issue of Molecular Cell, Benvegnù et al. (2017) report an unexpected phenomenon by which the E3 ligase mahogunin ring finger-1 (MGRN1) translocates to the nucleus in an age-dependent manner, revealing an intriguing mechanism that allows for an adaptive neuronal response to proteotoxic stress, often seen with aging.