Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition

Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function

Innovative solutions to sticky situations: Antiadhesive strategies for treating bacterial infections

ABSTRACT Bacterial adherence to host tissue is an essential process in pathogenesis, necessary for invasion and colonization and often required for the efficient delivery of toxins and other bacterial effectors. As existing treatment options for common bacterial infections dwindle, we find ourselves rapidly approaching a tipping point in our confrontation with antibiotic-resistant strains and in desperate need of new treatment options. Bacterial strains defective in adherence are typically avirulent and unable to cause infection in animal models. The importance of this initial binding event in the pathogenic cascade highlights its potential as a novel therapeutic target. This article seeks to highlight a variety of strategies being employed to treat and prevent infection by targeting the mechanisms of bacterial adhesion. Advancements in this area include the development of novel antivirulence therapies using small molecules, vaccines, and peptides to target a variety of bacterial infections. These therapies target bacterial adhesion through a number of mechanisms, including inhibition of pathogen receptor biogenesis, competition-based strategies with receptor and adhesin analogs, and the inhibition of binding through neutralizing antibodies. While this article is not an exhaustive description of every advancement in the field, we hope it will highlight several promising examples of the therapeutic potential of antiadhesive strategies.</jats:p

Intracellular nanosurgery and cell enucleation using a picosecond laser

Living cells are highly organized in space and time, which makes spatially and temporally confined manipulations an indispensable tool in cell biology. Laser-based nanosurgery is an elegant method that allows precise ablation of intracellular structures. Here, we show cutting of fluorescently labelled microtubules and mitotic spindles in fission yeast, performed with a picosecond laser coupled to a confocal microscope. Diverse effects from photo-bleaching to partial and complete breakage are obtained by varying the exposure time, while simultaneously imaging the structures of interest. Using this system we developed an efficient technique to generate enucleated cells without perturbing the distribution of other organelles. This enucleation method can be used to study the cytoskeleton in a nucleus-free environment, as well as the role of the nucleus in cell growth and a variety of cellular functions

Live cell multicolor imaging of lipid droplets with a new dye, LD540

A lipophilic dye based on the Bodipy fluorophore, LD540, was developed for microscopic imaging of lipid droplets. In contrast to previous lipid droplet dyes, it can spectrally be resolved from both green and red fluorophores allowing multicolor imaging in both fixed and living cells. Its improved specificity, brightness and photostability support live cell imaging, which was used to demonstrate by two-color imaging lipid droplet motility along microtubules

Dried-droplet probe preparation on AnchorChip targets for navigating the acquisition of matrix-assisted laser desorption/ionization time-of-flight spectra by fluorescence of matrix/analyte crystals.

We have developed a dried-droplet probe preparation method for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which uses AnchorChip targets and alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Upon drying of a matrix and analyte mixture on the AnchorChip, salts and low molecular weight contaminants were pooled at the hydrophilic metal anchor, whereas 10-50 microm matrix/peptide crystals firmly adhered at the surface of a hydrophobic polymer and the entire target could be subsequently washed by submerging it in 5% formic acid for 2-3 min. Epifluorescence microscopy suggested that peptides were completely co-localized with CHCA crystals at the AnchorChip surface. Fluorescent images of the probes were of good contrast and were background-free, compared with images taken by a video camera built into the ion source. CHCA/peptide crystals were easy to recognize at the surface and peptide mass maps were acquired from them without further adjustment of the position of the laser beam. These crystals were remarkably stable towards the laser depletion and almost no matrix-related ions were typically observed in the low m/z region of peptide mass maps. The sensitivity of the peptide mass mapping was at the low-femtomole level

Dried-droplet probe preparation on AnchorChip targets for navigating the acquisition of matrix-assisted laser desorption/ionization time-of-flight spectra by fluorescence of matrix/analyte crystals.

We have developed a dried-droplet probe preparation method for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which uses AnchorChip targets and alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. Upon drying of a matrix and analyte mixture on the AnchorChip, salts and low molecular weight contaminants were pooled at the hydrophilic metal anchor, whereas 10-50 microm matrix/peptide crystals firmly adhered at the surface of a hydrophobic polymer and the entire target could be subsequently washed by submerging it in 5% formic acid for 2-3 min. Epifluorescence microscopy suggested that peptides were completely co-localized with CHCA crystals at the AnchorChip surface. Fluorescent images of the probes were of good contrast and were background-free, compared with images taken by a video camera built into the ion source. CHCA/peptide crystals were easy to recognize at the surface and peptide mass maps were acquired from them without further adjustment of the position of the laser beam. These crystals were remarkably stable towards the laser depletion and almost no matrix-related ions were typically observed in the low m/z region of peptide mass maps. The sensitivity of the peptide mass mapping was at the low-femtomole level