95 research outputs found
PCNA appears in two populations of slow and fast diffusion with a constant ratio throughout S-phase in replicating mammalian cells
DNA replication is a fundamental cellular process that precedes cell division. Proliferating cell nuclear antigen (PCNA) is a central scaffold protein that orchestrates DNA replication by recruiting many factors essential for the replication machinery. We studied the mobility of PCNA in live mammalian cells using single-particle tracking in combination with photoactivated-localization microscopy (sptPALM) and found two populations. The first population which is only present in cells with active DNA replication, showed slow diffusion and was found to be located in replication foci. The second population showed fast diffusion, and represents the nucleoplasmic pool of unbound PCNA not involved in DNA replication. The ratio of these two populations remained constant throughout different stages of S-phase. A fraction of molecules in both populations showed spatially constrained mobility. We determined an exploration radius of ~100 nm for 13% of the slow-diffusing PCNA molecules, and of ~600 nm for 46% of the fast-diffusing PCNA molecules
Collagen organization within the cartilage of TRPV4(-/-) mice studied with two-photon microscopy and polarized second harmonic generation
The polymodal channel TRPV4 has been shown to regulate development and maintenance of cartilage. Here we investigate whether TRPV4 activity regulates the early deposition and structure of collagen matrix in the femoral head cartilage by comparing the 3D morphology and the sub-micrometer organization of the collagen matrix between wild type and TRPV4(-/-) mice pups four to five days old. Two-photon microscopy can be used to conduct label-free imaging of cartilage, as collagen generates a second harmonic signal (second harmonic generation [SHG]) under pulsed infrared excitation. In one set of measurements, we use circularly polarized laser light to reconstruct the 3D morphology of the femoral head cartilage and to measure the tissue thickness. Second, by rotating the direction of the linearly polarized light and using polarized SHG detection, we investigate the sub-micrometer orientation of collagen fibers in the cartilage. At this developmental stage, we cannot detect statistically significant differences between the two mice strains, although a tendency toward a more random orientation of collagen fibers and a higher thickness of the whole cartilage seems to characterize the TRPV4(-/-) mice. We discuss possible reasons for these observations
Highly resolved intravital striped-illumination microscopy of germinal centers
Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells - on the level of a few protein molecules in germinal centers
High-resolution intravital microscopy
Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector
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
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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
In vitro production of naked mole-rats' blastocysts from non-breeding females using in vitro maturation and intracytoplasmic sperm injection
The African naked mole-rat (Heterocephalus glaber) is an attractive model for cancer and aging research due to its peculiar biological traits, such as unusual long life span and resistance to cancer. The establishment of induced pluripotent stem cells (iPSCs) would be a useful tool for in vitro studies but, in this species, the reprogramming of somatic cells is problematic because of their stable epigenome. Therefore, an alternative approach is the derivation of embryonic stem cells from in vitro-produced embryos. In this study, immature oocytes, opportunistically retrieved from sexually inactive females, underwent first in vitro maturation (IVM) and then in vitro fertilization via piezo-intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultivated with two different approaches: (i) in an in vitro culture and (ii) in an isolated mouse oviduct organ culture system. The second approach led to the development of blastocysts, which were fixed and stained for further analysis
TSG101 associates with PARP1 and is essential for PARylation and DNA damage-induced NF-κB activation
In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-κB pathway, we identified scores of regulators, including tumor susceptibility gene TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKKγ signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-κB activation, TSG101-deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP inhibition. We also show that the loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment
PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step
The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA
Functional traits influence patterns in vegetative and reproductive plant phenology - a multi-botanical garden study
Phenology has emerged as key indicator of the biological impacts of climate change, yet the role of functional traits constraining variation in herbaceous species' phenology has received little attention. Botanical gardens are ideal places in which to investigate large numbers of species growing under common climate conditions. We ask whether interspecific variation in plant phenology is influenced by differences in functional traits. We recorded onset, end, duration and intensity of initial growth, leafing out, leaf senescence, flowering and fruiting for 212 species across five botanical gardens in Germany. We measured functional traits, including plant height, absolute and specific leaf area, leaf dry matter content, leaf carbon and nitrogen content and seed mass and accounted for species' relatedness. Closely related species showed greater similarities in timing of phenological events than expected by chance, but species' traits had a high degree of explanatory power, pointing to paramount importance of species' life-history strategies. Taller plants showed later timing of initial growth, and flowered, fruited and underwent leaf senescence later. Large-leaved species had shorter flowering and fruiting durations. Taller, large-leaved species differ in their phenology and are more competitive than smaller, small-leaved species. We assume climate warming will change plant communities' competitive hierarchies with consequences for biodiversity
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