Journal of cell science最新文献

The GLI1, GLI2 and GLI3 transcription factors mediate Hedgehog (Hh) signaling, which is crucial for bone development. During intramembranous ossification, mesenchymal stem cells (MSCs) are directly differentiated into osteoblasts. Under basal and Hh pathway-stimulated conditions, primary cilia play essential roles in proteolytic processing of GLI3 to its repressor form (GLI3R) and in activation of GLI2. Although previous studies in mice have suggested that Gli1 expression depends on GLI2 and GLI3, coordinated roles of GLI1, GLI2 and GLI3 in osteogenic differentiation are not fully understood at the cellular level. From the MSC line C3H10T1/2, we established Gli2-knockout (KO) and Gli3-KO cells, as well as constitutively GLI3R-producing (cGLI3R) cells, and expressed GLI1, GLI2 and GLI3 constructs in these cell lines. The results demonstrate at the cellular level that GLI2 and GLI3R counterregulate osteogenic differentiation via activation and repression of Gli1 expression, respectively; GLI3R, which results from GLI3 processing requiring protein kinase A-mediated phosphorylation, downregulates expression of Gli2 as well as Gli1; and GLI1 upregulates expression of Gli1 itself and Gli2, constituting a GLI1-GLI2 positive feedback loop.

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei and cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. To determine the composition of nuclear blebs, we compared the immunofluorescence intensity of DNA and lamin B in the main nucleus body to that in the nuclear bleb across cell types and perturbations. DNA density in the nuclear bleb was consistently decreased to about half that of the nuclear body whereas lamin B levels in the nuclear bleb varied widely. Partial wave spectroscopic (PWS) microscopy recapitulated the significantly decreased likelihood of high-density domains in the nuclear bleb versus body, and that it was independent of lamin B level. Time-lapse imaging into immunofluorescence revealed that decreased DNA density marked all nuclear blebs whereas decreased lamin B1 levels only occurred in blebs that had recently ruptured. Thus, decreased DNA density is a better marker of a nuclear bleb than lamin B level.

Wnt signalling is an essential signalling system in neurogenesis, with a crucial role in synaptic plasticity and neuronal survival, processes that are disrupted in Alzheimer's disease (AD). Within this network, the Wnt/β-catenin pathway has been studied for its neuroprotective role, and this is suppressed in AD. However, the involvement of the non-canonical Wnt-planar cell polarity (Wnt/PCP) pathway in AD remains to be determined. This study investigates the role of ROR2, a Wnt/PCP co-receptor, in synaptogenesis. We demonstrate that WNT5A-ROR2 signalling activates the JNK pathway, leading to synapse loss in mature neurons. This effect mirrors the synaptotoxic actions of Aβ1-42 and DKK1, which are elevated in AD. Notably, blocking ROR2 and JNK mitigates Aβ1-42 and DKK1-induced synapse loss, suggesting their dependence on ROR2. In induced pluripotent stem cell (iPSC)-derived cortical neurons carrying a PSEN1 mutation, known to increase the Aβ42/40 ratio, we observed increased WNT5A-ROR2 clustering and reduced numbers of synapses. Inhibiting ROR2 or JNK partially rescued synaptogenesis in these neurons. These findings suggest that, unlike the Wnt/β-catenin pathway, the Wnt/PCP-ROR2 signalling pathway can operate in a feedback loop with Aβ1-42 to enhance JNK signalling and contribute to synapse loss in AD.

Various N-terminal tags have often been used to identify the functions and localization of Rab small GTPases, but their impact on Rab proteins themselves has been poorly investigated. Here, we used a knockout (KO)-rescue approach to systematically evaluate the effect of N-terminal tagging of two Rabs, Rab10 and Rab27A, on RAB10-KO HeLa cells and Rab27A-deficient melanocytes (melan-ash cells), respectively. The results showed that all of the N-terminal-tagged Rab27A proteins mediated actin-based melanosome transport in the melan-ash cells, but none of the N-terminal-tagged Rab10 proteins fully rescued the defect in tubular endosome formation in RAB10-KO cells. Although the N-terminal-tagged Rab10 proteins had the ability to localize tubular endosomes in wild-type HeLa cells, they sometimes exhibited a dominant-negative effect on tubular endosome formation. We also found that a conserved lysine residue at amino acid position 3 (K3) in the Rab10 proteins of different species is required for tubular endosome formation. Thus, it will be important to determine whether other Rab isoforms with N-terminal tags behave similarly to their corresponding untagged isoforms by performing appropriate KO-rescue experiments in future studies.

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements and DNA damage. Nuclear blebs (i.e. herniations of the nuclear envelope) can be induced by (1) nuclear compression, (2) nuclear migration (e.g. cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated whether (1) packing domain organization is altered within nuclear blebs and (2) whether alteration in packing domain structure contributed to bleb formation. Using dual-partial wave spectroscopic microscopy, we show that chromatin-packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to what is seen in the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that chromatin within blebs is associated with a fragmented higher-order chromatin structure.

Pluripotent Stem Cells (PSCs) exhibit extraordinary differentiation potential and are thus highly valuable cellular model systems. However, while different PSC types corresponding to distinct stages of embryogenesis have been in common use, aspects of their cellular architecture and mechanobiology remain insufficiently understood. Here we investigated how the actin cytoskeleton is regulated in different pluripotency states. We observed a drastic reorganization during the transition from ground-state naïve mouse embryonic stem cells (mESCs) to converted prime epiblast stem cells (EpiSCs). mESCs are characterized by prominent actin-enriched cortical structures that contain cadherin-based cell-cell junctional components, despite not locating at cell-cell junctions. We termed these structures "Non-Junctional Cadherin Complexes (NJCC)" and showed that they are under low mechanical tension, depend on the ectodomain but not the cytoplasmic domain of E-cadherin, and exhibit minimal calcium dependence. Active Rac1 was identified as a negative regulator that promotes β-catenin dissociation and NJCC fragmentation. Our data suggests that NJCC may arise from the cis-association of E-cadherin ectodomain, with potential roles in ground-state pluripotency, and could serve as structural markers to distinguish heterogeneous population of pluripotent stem cells.

Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dynein's function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the size of ionotropic glutamate receptor (iGluRs) fields, and an increase in the amplitude and frequency of mEJPs. PI(4,5)P2 levels do not affect iGluR clustering, nor does dynein affect the levels of iGluR subunits at the NMJ. Our observations suggest a separate, transport independent function for dynein in iGluR cluster organization. Based on the close apposition of dynein punctae to the iGluR fields, we speculate that dynein at the postsynaptic membrane contributes to the organization of the receptor fields, hence ensuring proper synaptic transmission.

Glycosaminoglycans (GAGs), as animal polysaccharides, are linked to proteins to form various types of proteoglycans. Bacterial GAG lyases are not only essential enzymes that spoilage bacteria use for the degradation of GAGs, but also valuable tools for investigating the biological function and potential therapeutic applications of GAGs. The ongoing discovery and characterization of novel GAG lyases has identified an increasing number of lyases suitable for functional studies and other applications involving GAGs, which include oligosaccharide sequencing, detection and removal of specific glycan chains, clinical drug development and the design of novel biomaterials and sensors, some of which have not yet been comprehensively summarized. GAG lyases can be classified into hyaluronate lyases, chondroitinases and heparinases based on their substrate spectra, and their functional applications are mainly determined by their substrates, with different lyases exhibiting differing substrate selectivity and preferences. It is thus necessary to understand the properties of the available enzymes to determine strategies for their functional application. Building on previous studies and reviews, this Review highlights small yet crucial differences among or within the various GAG lyases to aid in optimizing their use in future studies. To clarify ideas and strategies for further research, we also discuss several traditional and novel applications of GAG lyases.

The Rab11-Rabin8-Rab8 ciliogenesis complex regulates the expansion of cilia-derived light-sensing organelles, the rod outer segments, via post-Golgi rhodopsin transport carriers (RTCs). Rabin8 (also known as RAB3IP), an effector of Rab11 proteins and a nucleotide exchange factor (GEF) for Rab8 proteins, is phosphorylated at S272 by NDR2 kinase (also known as STK38L), the canine early retinal degeneration (erd) gene product linked to the human ciliopathy Leber congenital amaurosis (LCA). Here, we define the step at which NDR2 phosphorylates Rabin8 and regulates Rab11-to-Rab8 succession in Xenopus laevis transgenic rod photoreceptors expressing human GFP-Rabin8 and its mutants. GFP-Rabin8 accumulated with endogenous Rabin8 at the Golgi-apposed exit sites (GESs), also known as the trans-Golgi network (TGN). Rabin8 mutants deficient in Rab11 binding prevented membrane association of GFP-Rabin8. GFP-Rabin8 and NDR2 kinase both interacted with the RTC-associated R-SNARE VAMP7 at the trans-Golgi and the GESs. Here, GFP-Rabin8 and the phosphomimetic GFP-Rabin8-S272E integrated into RTCs, which were subsequently functionalized by Rabin8 Rab8 GEF activity. Non-phosphorylatable GFP-Rabin8-S272A caused significant GES enlargement and deformation, possibly leading to unconventional membrane advancement toward the cilium, bypassing RTCs. Rabin8 phosphorylation loss due to an NDR2 gene disruption thereby likely causes dysfunctional rhodopsin Golgi-to-cilia trafficking underlying retinal degeneration and early-onset blindness.