Biology subjects

Brandenburg, S.

Publications and source records attributed to Brandenburg, S..

3 recordsLinked to original sources

bioRxiv2025

A versatile microfluidics platform for enhanced multi-target super-resolution microscopy

DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) is a powerful variant of single-molecule localization microscopy (SMLM) that overcomes the limitations of photobleaching, offers flexible fluorophore selection, and enables fine control of imaging parameters through tunable on- and off-binding kinetics. Its most distinctive feature is the capacity for multiplexing, which is achieved through a process known as Exchange-PAINT. This technique involves assigning orthogonal DNA strands to different targets within a sample and then sequentially adding and removing complementary imager strands that are specific to only one target at a time. However, manual Exchange-PAINT workflows are often inefficient, prone to drift and variability, and lack reproducibility. Here, we introduce a custom compressed-air-driven microfluidics system specifically designed for multiplexed SMLM. Featuring a stackable and modular design that is, in principle, not limited by the number of channels, the system ensures robust, reproducible, and material-efficient buffer exchange with minimal dead volume. It operates in both manual and automated modes and can be readily adapted to a wide range of commercial and custom microscopes, including wide-field, confocal, STED, and MINFLUX platforms. We demonstrate robust 5-plex Exchange-PAINT imaging in cancerous U2OS cells and, importantly, we establish multiplexed nanoscale imaging in fragile primary cardiomyocytes. These applications highlight the unique power of our platform to extend super-resolution multiplexing into physiologically relevant systems, thereby opening new avenues for biomedical research.

biophysics↗

bioRxiv2024

Development of a protein synthesis network at the sarco/endoplasmic reticulum in adult cardiac myocytes

IntroductionThe endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins, which constitute a large fraction of the total protein output of a mammalian cell. Striated muscle cells contain a specialized membrane system known as the sarcoplasmic reticulum (SR) that controls calcium homeoastasis and contraction, however the biochemical and physiological relationship between the ER and SR and how both compartments participate in protein synthesis remains incompletely understood. MethodsProtein quantification and imaging of selected marker proteins for ER and SR functions was performed to characterize the relationship of the ER and SR and its individual involvement in protein synthesis in neonatal and adult cardiac myocytes. Superresolution microscopy was used to examine the interaction of ribosomes and the SR in adult cardiac myocytes. ResultsQuantification of ER and SR-associated proteins of isolated ventricular cardiac myocytes showed that relative expression of ER/SR resident protein quality elements, as well as relative ribosome levels are decreased in adult cells, whereas SR-associated Ca2+ handling proteins increase. Immunocytoflourescence revealed that the membrane compartment that exists in early postpartum resembles mostly the ER and decreases in postnatal development. The SR is the main membrane network that exists in the adult cardiac myocytes, replacing the ER, in all but the perinuclear region. Immunocytoflourescence staining further indicated that both networks perform overlapping but distinct, specialized functions, such as localization of excitation-contraction coupling exclusively to the SR or initiation of secretion via the classical secretory pathway mainly from the ER. Ribosomes and mRNA were localized both in close proximity to the ER and the SR of adult ventricular cardiac myocytes. Superresolution microscopy confirmed that both the ER as well as the developed SR bind ribosomes and are direct sites of protein synthesis and protein homeostasis in adult cardiac myocytes. ConclusionOur findings suggest molecular differentiation and structural organization of the ER/SR in cardiac muscle development, resulting in the development of a protein synthesis network at the sarco/endoplasmic reticulum in adult cardiac myocytes.

cell biology↗

bioRxiv2020

CDK4/6 inhibition promotes senescent states with restricted secretory phenotype and reduced toxicity

Cellular senescence is a state of stable growth arrest that acts as a tumor suppressive mechanism. Several anti-cancer interventions function partly by inducing malignant cells into senescence. However, because of systemic administration and lack of specificity, anti-cancer treatments are associated with premature senescence of various non-malignant cells. Therapy-induced non-malignant senescent cells can have profound detrimental pro-tumorigenic and pro-disease functions via activation of a pro-inflammatory and NF-{kappa}B-mediated secretory phenotype (SASP). Inhibitors of the cyclin-dependent kinases 4/6 (CDK4/6i) has recently shown to have potent cytostatic effects with reduced toxicities. Here, we show that CDK4/6i lead non-malignant cells to a senescent state that lacks the pro-inflammatory and NF-{kappa}B-associated SASP. Interestingly, CDK4/6i-induced senescence overexpressed a number of genes encoding for secreted proteins, which we show being dependent on p53 transcriptional activity. CDK4/6i-induced p16+ senescent cells with a p53-associated (PASP), but not NF-{kappa}B-associated (NASP), secretory phenotype do not exert detrimental and pro-tumorigenic functions, but still retain the capacity to induce paracrine senescence and undergo clearance in vivo. Our data suggest that that senescent cells with a PASP but without a NASP may be well-tolerated and may represent a less toxic outcome for cancer interventions.

cell biology↗