PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery

Balancing WNT signalling in early forebrain development: The role of LRP4 as a modulator of LRP6 function

The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling

Balancing WNT signalling in early forebrain development: The role of LRP4 as a modulator of LRP6 function

The specification of the forebrain relies on the precise regulation of WNT/ß-catenin signalling to support neuronal progenitor cell expansion, patterning, and morphogenesis. Imbalances in WNT signalling activity in the early neuroepithelium lead to congenital disorders, such as neural tube defects (NTDs). LDL receptor-related protein (LRP) family members, including the well-studied receptors LRP5 and LRP6, play critical roles in modulating WNT signalling capacity through tightly regulated interactions with their co-receptor Frizzled, WNT ligands, inhibitors and intracellular WNT pathway components. However, little is known about the function of LRP4 as a potential modulator of WNT signalling in the central nervous system. In this study, we investigated the role of LRP4 in the regulation of WNT signalling during early mouse forebrain development. Our results demonstrate that LRP4 can modulate LRP5- and LRP6-mediated WNT signalling in the developing forebrain prior to the onset of neurogenesis at embryonic stage 9.5 and is therefore essential for accurate neural tube morphogenesis. Specifically, LRP4 functions as a genetic modifier for impaired mitotic activity and forebrain hypoplasia, but not for NTDs in LRP6-deficient mutants. In vivo and in vitro data provide evidence that LRP4 is a key player in fine-tuning WNT signalling capacity and mitotic activity of mouse neuronal progenitors and of human retinal pigment epithelial (hTERT RPE-1) cells. Our data demonstrate the crucial roles of LRP4 and LRP6 in regulating WNT signalling and forebrain development and highlight the need to consider the interaction between different signalling pathways to understand the underlying mechanisms of disease. The findings have significant implications for our mechanistic understanding of how LRPs participate in controlling WNT signalling

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts

A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease

BACKGROUND: Alzheimer's disease (AD) is characterized by the intra- and extracellular accumulation of amyloid-β (Aβ) peptides. How Aβ aggregates perturb the proteome in brains of patients and AD transgenic mouse models, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive Aβ aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously. METHODS: We quantified progressive Aβ aggregation in hippocampus and cortex of 5xFAD mice and controls with immunohistochemistry and membrane filter assays. Protein changes in different mouse tissues were analyzed by MS-based proteomics using label-free quantification; resulting MS data were processed using an established pipeline. Results were contrasted with existing proteomic data sets from postmortem AD patient brains. Finally, abundance changes in the candidate marker Arl8b were validated in cerebrospinal fluid (CSF) from AD patients and controls using ELISAs. RESULTS: Experiments revealed faster accumulation of Aβ42 peptides in hippocampus than in cortex of 5xFAD mice, with more protein abundance changes in hippocampus, indicating that Aβ42 aggregate deposition is associated with brain region-specific proteome perturbations. Generating time-resolved data sets, we defined Aβ aggregate-correlated and anticorrelated proteome changes, a fraction of which was conserved in postmortem AD patient brain tissue, suggesting that proteome changes in 5xFAD mice mimic disease-relevant changes in human AD. We detected a positive correlation between Aβ42 aggregate deposition in the hippocampus of 5xFAD mice and the abundance of the lysosome-associated small GTPase Arl8b, which accumulated together with axonal lysosomal membranes in close proximity of extracellular Aβ plaques in 5xFAD brains. Abnormal aggregation of Arl8b was observed in human AD brain tissue. Arl8b protein levels were significantly increased in CSF of AD patients. CONCLUSIONS: We report a comprehensive biochemical and proteomic investigation of hippocampal and cortical brain tissue derived from 5xFAD transgenic mice, providing a valuable resource to the neuroscientific community. We identified Arl8b, with significant abundance changes in 5xFAD and AD patient brains. Arl8b might enable the measurement of progressive lysosome accumulation in AD patients and have clinical utility as a candidate biomarker

DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters.

The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility