Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays

Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating

Journal Staff

Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.Funding Agencies|Swedish Research Council for Medicine and Health|2007-34832009-66492010-3045|</p

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells. © The Author(s). Published by IOP Publishing Ltd

Disruption of hypoxia-inducible fatty acid binding protein 7 induces beige fat-like differentiation and thermogenesis in breast cancer cells

Background Humans produce heat through non-shivering thermogenesis, a metabolic process that occurs in inducible beige adipocytes expressing uncoupling protein 1 (UCP1). UCP1 dissipates the proton gradient of the mitochondrial inner membrane and converts that energy into heat. It is unclear whether cancer cells can exhibit autonomous thermogenesis. Previously, we found that the knockdown of hypoxia-inducible fatty acid binding protein 7 (FABP7) increased reactive oxygen species (ROS) in breast cancer cells. ROS are known to induce beige adipocyte differentiation. Methods We investigated the association of tumor hypoxia, FABP7, and UCP1 across breast cancer patients using METABRIC and TCGA data sets. Furthermore, using a breast cancer cell line, HCC1806, we tested the effect of FABP7 knockdown on cellular physiology including thermogenesis. Results We found a strong mutual exclusivity of FABP7 and UCP1 expression both in METABRIC and in TCGA, indicating major metabolic phenotypic differences. FABP7 was preferentially distributed in poorly differentiated-, estrogen receptor (ER) negative tumors. In contrast, UCP1 was highly expressed in normal ducts and well-differentiated-, ER positive-, less hypoxic tumors. In the cell line-based experiments, UCP1 and its transcriptional regulators were upregulated upon FABP7 knockdown. UCP1 was induced in about 20% of cancer cells, and the effect was increased further in hypoxia. UCP1 depolarized mitochondrial membranes at the site of expression. UCP1 induction was associated with the increase in proton leak, glycolysis, and maximal respiration, mimicking the typical energy profile of beige adipocytes. Most importantly, UCP1 induction elevated cancer cell temperature associated with increased vulnerability to hypoxia and gamma-irradiation. Conclusions We demonstrated that breast cancer cells can undergo thermogenesis through UCP1 induction. Disrupting FABP7-mediated fatty acid metabolism can unlock UCP1-mediated thermogenesis, potentially making it possible to develop therapies to target thermogenesis. Further study would be warranted to investigate the effect of rise in temperature of cancer cells on patients' outcomes and the relationship to other metabolic pathways

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes

Intracellular Photophysics of an Osmium Complex bearing an Oligothiophene Extended Ligand

This contribution describes the excited-state properties of an Osmium-complex when taken up into human cells. The complex 1 [Os(bpy)2(IP-4T)](PF6)2 with bpy=2,2′-bipyridine and IP-4T=2-{5′-[3′,4′-diethyl-(2,2′-bithien-5-yl)]-3,4-diethyl-2,2′-bithiophene}imidazo[4,5-f][1,10]phenanthroline) can be discussed as a candidate for photodynamic therapy in the biological red/NIR window. The complex is taken up by MCF7 cells and localizes rather homogeneously within in the cytoplasm. To detail the sub-ns photophysics of 1, comparative transient absorption measurements were carried out in different solvents to derive a model of the photoinduced processes. Key to rationalize the excited-state relaxation is a long-lived 3ILCT state associated with the oligothiophene chain. This model was then tested with the complex internalized into MCF7 cells, since the intracellular environment has long been suspected to take big influence on the excited state properties. In our study of 1 in cells, we were able to show that, though the overall model remained the same, the excited-state dynamics are affected strongly by the intracellular environment. Our study represents the first in depth correlation towards ex-vivo and in vivo ultrafast spectroscopy for a possible photodrug. © 2020 The Authors. Published by Wiley-VCH Gmb