Search bioRxiv⌕ Search

Biology subjects

Zipfel, W. R.

Publications and source records attributed to Zipfel, W. R..

7 recordsLinked to original sources

Polarization Increases Nuclear Stiffness in Macrophages Despite Reduction in Lamin A/C Levels

Macrophages are innate immune cells contributing to tissue homeostasis and various pathologies. Signals from their environment can lead macrophages to adapt distinct functional phenotypes, a process called polarization. Because macrophages have been previously shown to degrade the nuclear envelope proteins lamin A/C upon pro-inflammatory polarization, and lamins are considered key determinants of nuclear deformability, we aimed to address the effect of pro-inflammatory stimulation on nuclear mechanics. We present the surprising finding that polarized bone marrow-derived macrophages have less deformable nuclei than unpolarized macrophages, despite their reduced lamin A/C levels. Furthermore, pro-inflammatory macrophages exhibited altered chromatin dynamics relative to unpolarized macrophages, including redistribution of trimethylated histone H3K9 (H3K9me3) from the nuclear periphery to the interior and increased chromatin compaction. Our findings suggest a model in which pro-inflammatory stimulation of macrophages induces chromatin changes that drive nuclear stiffening, and that in these cells, chromatin, rather than the nuclear lamina, is the major driver for resisting nuclear deformation. These findings may have functional relevance for the physiological function of polarized macrophages, as the mechanical properties of the nucleus can influence how these cells adapt and respond to their environments in the context of cell migration or inflammatory disease pathologies.

cell biology↗

Assessing cellular metabolic dynamics with NAD(P)H fluorescence polarization imaging

Altered metabolism enables adaptive advantages for cancer, driving the need for improved methods for non-invasive long-term monitoring of cellular metabolism from organelle to population level. Here we present two-photon steady-state fluorescence polarization ratiometric microscopy (FPRM), a label-free imaging method that uses nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence as a functional readout of cellular metabolism. The method is simple to implement and operates an order of magnitude faster than the NAD(P)H-fluorescence lifetime imaging microscopy (FLIM) imaging modality, reducing cytotoxic stress while providing long-term monitoring capacity. FPRM enables high-resolution dynamic tracking of NAD(P)H signals with subcellular details and we have established a set of instrument-independent ratiometric parameters that correlates NAD(P)H signals with metabolic status during pharmaceutical and environmental perturbations. We further integrated FPRM readouts with other parameters such as cell shape and migration on 2D and 3D collagen matrices, demonstrating the techniques versatility across bioengineered platforms for cancer metabolism research.

biophysics↗

Spatial Mapping of Mobile Genetic Elements and their Cognate Hosts in Complex Microbiomes

The frequent exchange of mobile genetic elements (MGEs) between bacteria accelerates the spread of functional traits, including antimicrobial resistance, within the human microbiome. Yet, progress in understanding these intricate processes has been hindered by the lack of tools to map the spatial spread of MGEs in complex microbial communities, and to associate MGEs to their bacterial hosts. To overcome this challenge, we present an imaging approach that pairs single molecule DNA Fluorescence In Situ Hybridization (FISH) with multiplexed ribosomal RNA FISH, thereby enabling the simultaneous visualization of both MGEs and host bacterial taxa. We used this methodology to spatially map bacteriophage and antimicrobial resistance (AMR) plasmids in human oral biofilms, and we studied the heterogeneity in their spatial distributions and demonstrated the ability to identify their host taxa. Our data revealed distinct clusters of both AMR plasmids and prophage, coinciding with densely packed regions of host bacteria in the biofilm. These results suggest the existence of specialized niches that maintain MGEs within the community, possibly acting as local hotspots for horizontal gene transfer. The methods introduced here can help advance the study of MGE ecology and address pressing questions regarding antimicrobial resistance and phage therapy.

microbiology↗

Mucins form a nanoscale material barrier against immune cell attack

The cancer cell glycocalyx serves as a major line of defense against immune surveillance. However, how specific physical properties of the glycocalyx contribute to immune evasion and how these properties are regulated are not well understood. Here, we uncover how the surface density, glycosylation, and crosslinking of cancer-associated mucins contribute to the nanoscale material thickness of the glycocalyx, and further analyze the effect of the glycocalyx thickness on resistance to effector cell attack. Natural Killer (NK) cell-mediated cytotoxicity exhibits a near perfect inverse correlation with the glycocalyx thickness of target cells regardless of the specific glycan structures present. NK cells expressing a chimeric antigen receptor (CAR) have an enhanced ability to breach the glycocalyx and kill target cells. Equipping the NK cell surface with a mucin-digesting enzyme also improves killing with a performance enhancement that rivals or exceeds CARs in some cases. Together, our results provide new considerations for improving cancer immunotherapies.

biophysics↗

Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents.

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins. KEY POINTSO_LIA simple to use RNA aptamer method for super-resolution STORM and PAINT imaging in cells expressing common protein tags. C_LIO_LIThe method has a number of unique advantages for live cell imaging at the nanometer scale. C_LIO_LIProvides a means to quantify the number of proteins being trafficked on the plasma membrane. C_LI

biophysics↗

Litmus-Body: a Molecularly Targeted Sensor for Cell-Surface pH Measurements

Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the molecular specificity required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-mouse IgG nanobody is capable of molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long-Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488-nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the physiological regime of pH. The sensor was further validated for use on live cells, shown to be specific to the protein of interest, and was able to successfully recapitulate the numerous pH changes along the endocytic pathway.

cell biology↗

High-speed device synchronization in optical microscopy with an open-source hardware control platform

Recent advances in fluorescence microscopy have enabled the visualization of subcellular structures at unprecedented resolution. However, the complexity of state-of-the-art microscopes has increased considerably, often requiring the precise control and synchronization of multiple peripheral devices at high speeds. Drawing inspiration from open-source prototyping systems, like the Arduino, we describe the development of a new control platform that adopts the best features of these systems - affordability, facile programmability, and flexible connectivity - but with the scientific-grade inputs and outputs (I/O) and built-in routines that are necessary to control peripherals in advanced microscopy applications. Notably, our platform includes waveform generators and I/O for point-and azimuthal-scanning of excitation in laser-based applications. As a proof of concept, we show how the integration of waveform generation, multiplexed analog outputs, and native hardware triggers into a single central hub provides a versatile platform for performing fast circle-scanning acquisitions, including ring scanning-angle interference microscopy (SAIM), total internal reflection fluorescence (TIRF and ring TIRF) microscopy, and multiangle TIRF (MA-TIRF). We also demonstrate how the low communication latency of our hardware platform can reduce image intensity and reconstruction artifacts arising from synchronization errors produced by software control. Our complete platform, including hardware design files, firmware, API, software, and all associated source code, will be hosted for community-based development and collaboration.

biophysics↗