Melike Lakadamyali

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

A DNA origami platform for quantifying protein copy number in super-resolution

Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy.

3D motion of vesicles along microtubules helps them to circumvent obstacles in cells

ABSTRACT Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the separation of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them. Highlighted Article: We develop a new method to track vesicle dynamics in three dimensions and show that vesicles change their position while actively transporting along microtubules in living cells to overcome obstacles.

Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis

Adhesion to the extracellular matrix persists during mitosis in most cell types. However, while classical adhesion complexes, such as focal adhesions, do and must disassemble to enable mitotic rounding, the mechanisms of residual mitotic cell–extracellular matrix adhesion remain undefined. Here, we identify ‘reticular adhesions’, a class of adhesion complex that is mediated by integrin αvβ5, formed during interphase, and preserved at cell–extracellular matrix attachment sites throughout cell division. Consistent with this role, integrin β5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Reticular adhesions are morphologically and dynamically distinct from classical focal adhesions. Mass spectrometry defines their unique composition, enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding proteins but lacking virtually all consensus adhesome components. Indeed, reticular adhesions are promoted by PtdIns(4,5)P2, and form independently of talin and F-actin. The distinct characteristics of reticular adhesions provide a solution to the problem of maintaining cell–extracellular matrix attachment during mitotic rounding and division.Lock et al. identify reticular adhesion complexes that maintain cell–extracellular-matrix attachments during cell division. Reticular adhesions transmit spatial memory between cell generations, mediated by αvβ5 integrin and PtdIns(4,5)P2.

Reticular adhesions: A new class of adhesion complex that mediates cell-matrix attachment during mitosis

Adhesion to the extracellular matrix (ECM) persists during mitosis in most cell types. Yet, classical adhesion complexes (ACs), such as focal adhesions and focal complexes, do and must disassemble to enable cytoskeletal rearrangements associated with mitotic rounding. Given this paradox, mechanisms of mitotic cell-ECM adhesion remain undefined. Here, we identify ‘reticular adhesions’, a new class of AC that is mediated by integrin αvβ5, formed during interphase and preserved at cell-ECM attachment sites throughout cell division. Consistent with this role, integrin β5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Quantitative imaging reveals reticular adhesions to be both morphologically and dynamically distinct from classic focal adhesions, while mass spectrometry defines their unique composition; lacking virtually all consensus adhesome components. Indeed, remarkably, reticular adhesions are functionally independent of both talin and F-actin, yet are promoted by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). Overall, the distinct characteristics of reticular adhesions provide a unique solution to the problem of maintaining cell-ECM attachment during mitotic rounding and division.

TiO2 Nanoparticle-Induced Oxidation of the Plasma Membrane: Importance of the Protein Corona

Titanium dioxide (TiO2) nanoparticles, used as pigments and photocatalysts, are widely present in modern society. Inhalation or ingestion of these nanoparticles can lead to cellular-level interactions. We examined the very first step in this cellular interaction, the effect of TiO2 nanoparticles on the lipids of the plasma membrane. Within 12 h of TiO2 nanoparticle exposure, the lipids of the plasma membrane were oxidized, determined with a malondialdehyde assay. Lipid peroxidation was inhibited by surface passivation of the TiO2 nanoparticles, incubation with an antioxidant (Trolox), and the presence of serum proteins in solution. Subsequent experiments determined that serum proteins adsorbed on the surface of the TiO2 nanoparticles, forming a protein corona, inhibit lipid peroxidation. Super-resolution fluorescence microscopy showed that these serum proteins were clustered on the nanoparticle surface. These protein clusters slow lipid peroxidation, but by 24 h, the level of lipid peroxidation is similar, independent of the protein corona or free serum proteins. Additionally, over 24 h, this corona of proteins was displaced from the nanoparticle surface by free proteins in solution. Overall, these experiments provide the first mechanistic investigation of plasma membrane oxidation by TiO2 nanoparticles, in the absence of UV light and as a function of the protein corona, approximating a physiological environment.

Super resolution imaging of chromatin in pluripotency, differentiation, and reprogramming

Chromatin fiber organization is essential for gene function in all cell types. Moreover it helps to determine cell fate in embryonic/adult stem cells and in somatic cells undergoing reprogramming to pluripotency. Until now the diffraction limit of light has limited the inspection of the chromatin fiber organization to a level sufficient to understand how it impacts gene function. The development of advanced microscopy methods, such as single molecule localization microscopy, has largely opened a new field of research providing us with the tools to visualize and quantitatively analyze chromatin fiber organization and thus gene activity at nanoscale resolution in single cells.

Quantifying motor protein copy number in super-resolution using an imaging invariant calibration

Motor proteins are nanoscale machines that convert the energy of ATP hydrolysis into the mechanical motion of walking along cytoskeletal filaments. In doing so, they transport organelles and help maintain sub-cellular organization. We previously developed a DNA origami-based calibration approach to extract protein copy number from super-resolution images. Using this approach, we show here that the retrograde motor protein dynein is mostly present as a single motor in the cytosol, whereas a small population of dynein along the microtubule cytoskeleton forms higher-order multimers organized into nano-sized domains. We further demonstrate, using dynein as a test sample, that the DNA origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any GFP-fused protein of interest. Our results have implications for motor coordination during intracellular trafficking as well as for using super-resolution as a quantitative method to determine protein copy number at the nanoscale level.

(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes

Abstract CRISPR/dCas9-based labeling has allowed direct visualization of genomic regions in living cells. However, poor labeling efficiency and signal-to-background ratio have limited its application to visualize genome organization using super-resolution microscopy. We developed (Po)STAC (Polycistronic SunTAg modified CRISPR) by combining CRISPR/dCas9 with SunTag labeling and polycistronic vectors. (Po)STAC enhances both labeling efficiency and fluorescence signal detected from labeled loci enabling live cell imaging as well as super-resolution fixed-cell imaging of multiple genes with high spatiotemporal resolution.