Atlas of Science

Tag: fluorescence microscopy

Method to measure calcium signaling in nanometer-sized primary cilia

Cilia are slender microtubule-based organelles that protrude from the apical membrane in most adherent cells. Studies looking into the mechanosensory function of the primary cilium have proved it to be an essential cellular organelle. When exposed to fluid flow, the flexible cilia bend and induces an intracellular calcium (Ca²⁺) influx followed by a Ca²⁺ increase throughout the cell. The initial influx has been attributed to the mechanosensory complex of polycystins in the ciliary membrane. Ca²⁺ are essential in biological systems as a secondary messenger and are involved in various function like regulating vascular tone, neurotransmitter release, muscle contraction, immune responses among many others. Thus, the ciliary Ca²⁺ influx in response to fluid flow can be a functional readout of cilia function. Technologies to study and measure functional ciliary Ca²⁺ changes are critical to understanding the changes occurring due to dysfunctional cilia. Over 20 ciliopathies, a spectrum of chronic multi-system disorders, have been identified which further bolsters the need to study the alterations in ciliary Ca²⁺ signaling in a live cell model.

Fig. 1. Single-live cell and cilia imaging with 5HT6-mCherry-G-GECO1.0. The images show (from left to right column) DIC used for tracking a cilium, mCherry fluorescent ciliary marker, the Ca²⁺ sensitive GECO1.0 and an EGFP/mCherry ratio pseudocolored to show Ca²⁺ levels. When fluid flow is applied (time series from top to bottom), the cilium bends inducing a Ca2+ increase in both the cytoplasm and cilioplasm.
Adapted with permission from Pala, R., Mohieldin, A. M., Shamloo, K., Sherpa, R. T., Kathem, S. H., Zhou, J., et al. Personalized nanotherapy by specifically targeting cell organelles to improve vascular hypertension. Nano Letters, 19, 904–914. Copyright 2018 American Chemical Society.

Among the key challenges to study cilia in living cells are their minute size and orientation. The primary cilia have a diameter around 200nm and unfortunately assumes a perpendicular orientation in relation to the cell monolayer. This limits the option to visualize the cilium together with the cytoplasm since they are at different planes of view. Secondly, traditional Ca²⁺ indicators fail to reach the cilioplasm due to the selective exclusion of most cellular components and exogenous compounds. To overcome this challenge, targeting modules have been utilized to transport cargo into the cilia. As an example, 5HT6-mCherry-G-GECO1.0 contains a cilium-targeting sequence derived from 5HT6, a serotonin receptor (Fig. 1). In 5HT6-mCherry-G-GECO1.0, the Ca²⁺ sensor G-GECO1.0 changes the fluorescence intensity of GFP with Ca²⁺ levels. In addition, the constant mCherry fluorescent marker is independent of Ca²⁺ flux serving as an aid in the visualization of ciliary movement, correction of artifacts, and ratiometric analysis of the data. There are a versatile number of calcium indicators and the choice is strictly determined by the needs of the researcher after evaluating key characteristics like the dynamic range, affinity (Kd) of the indicator for Ca²⁺, response kinetics, and targeting ability.

Fig. 2. Microwire with cells in a perfusion chamber. (A) In the diagram, the gasket is placed on the imaging dish in a manner to orient the microwire parallel to flow. Using lower magnifications, we can observe cells growing on the wire edge. At a higher magnification, the cilium protruding outward should become even clearer. We focus on a single cell and capture Ca²⁺ data in response to fluid flow. (B) Schematic of the experimental setup with stopcock valves to load and unload buffers into circulation.

In our experimental setup, cells are seeded on a microwire coated with collagen and then transfected with the 5HT6-mCherry-G-GECO1.0 construct (Fig. 2A). Proper microscope setup capable of rapid acquisition of GFP and mCherry signals at a speed compatible with the needs of the researcher is required. After the imaging chamber is set up, baseline measurement of the resting Ca²⁺ is acquired for 5 minutes followed by the introduction of flow with a peristaltic pump. The GFP intensity as a function of time will show an increase in signal intensity upon initiation of fluid flow. Depending on the experimental needs the buffers or treatments can be implemented using stopcock valves to introduce new perfusate and simultaneously eliminate the initial circulating buffer (Fig. 2B). In each experiment, minimum fluorescence intensity is measured during a Ca^2+-free solution containing EGTA and ionomycin. The treatment with ionomycin/EGTA causes a transient increase due to the extracellular Ca²⁺ entry from the ionomycin-induced pore formation followed by a slow decline in Ca²⁺ signal due to the Ca²⁺ chelator EGTA. In contrast, a buffer of Ca²⁺ (10mM) is introduced to obtain a maximum signal. These steps ensure that the sensor maintains the dynamic range (or Kd) and is responding to the ambient Ca²⁺ concentration.

Rinzhin T. Sherpa, Surya M. Nauli
Department of Biomedical & Pharmaceutical Sciences, Chapman University, Irvine, CA 92618, USA
Department of Medicine, Division of Nephrology, University of California Irvine, Irvine, CA 92697, USA
Department of Pharmacology, University of Nevada, Reno, NV 89557, USA

Publication

Measurement of cytoplasmic and cilioplasmic calcium in a single living cell
Rinzhin T Sherpa, Rajasekharreddy Pala, Ashraf M Mohieldin, Surya M Nauli
Methods Cell Biol. 2019

May 28, 2021
kINPen09 plasma treatment leads to antimicrobial effects against Candida albicans biofilms

Microbial communities are an increasing problem in medicine but also in industry. Thus, an efficient and rapid removal of biofilms from surfaces is becoming increasingly important. With the aid of the kINPen09, a radiofrequency plasma jet (RFPJ) instrument, decisive new findings on the effects of plasma on Candida albicans (strain SC5314) biofilms could be obtained. This work showed that the inactivation of biofilms takes place mainly on the bottom, which in turn offers new possibilities for the removal of biofilms by other strategies, e.g., mechanical treatment. This result demonstrated that non-thermal atmospheric pressure plasma is well suited for biofilm decontamination.

Fig. 1. Fluorescence microscopy of plasma-treated biofilms.
(A) Control; (B to F) plasma treatment for 30 s (B), 60 s (C), 120 s (D), 180 s (E), and 300 s (F). The biofilms were stained with SYTO9, showing green fluorescence (living cells), and propidium iodide, showing red fluorescence (dead cells).

In order to avoid external stresses, microorganisms are predominantly organized in biofilms. These biofilms have much higher resistance to external stresses like antibiotics or disinfections compared to their planktonic counterparts. This work showed that the non-thermal atmospheric pressure plasma treatment of C. albicans biofilms with the kINPen09 leads to a susceptibility in terms of growth, survival and cell viability. Morphological analysis of plasma-treated biofilms using atomic force microscopy supported the indications for lethal plasma effects concomitant with membrane disruptions and the loss of intracellular fluid. Fluorescence microscopy and confocal laser-scanning microscopy (CLSM) also provided the meaningful evidence that the biofilms were killed by the treatment predominantly on the bottom, where they adhere to the overgrown surface.

Fig. 2. Atomic-force microscopy images of the plasma-treated biofilms.
The left panels show topographical images from the top of the biofilm layer, and the right panels show error signal images of the same section. (A) Control; (B and C) plasma treatment times of 120 s (B) and 300 s (C). The images were taken in contact mode with a cantilever spring constant (k) of 0.1 to 0.6 N/m2 and a frequency of 0.4 Hz, the set point at 8 N/m2 , and an area of 20 m2.

In addition, CFU counts, and fluorescence- and XTT microplate -assays clearly demonstrated a strong negative effect on biofilm cells. These results are particularly significant, as in many industrial processes the decisive factor of decontamination is the biofilm detachment from the overgrown surface. In the course of the flushing processes, the detached biofilm mass is then removed from the facilities, whereby it is not relevant for the process whether the detached biofilm mass is completely dead.

O. Handorf, T. Weihe, S. Bekeschus, A. C. Graf, U. Schnabel, K. Riedel, J. Ehlbeck
Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
Ernst-Moritz-Arndt University, Microbial Physiology and Molecular Biology, Felix-Hausdorff-Str. 8, 17489 Greifswald, Germany

Publication

Nonthermal Plasma Jet Treatment Negatively Affects the Viability and Structure of Candida albicans SC5314 Biofilms.
Handorf O, Weihe T, Bekeschus S, Graf AC, Schnabel U, Riedel K, Ehlbeck J
Appl Environ Microbiol. 2018 Oct 17

January 7, 2020
Visualizing transcription factor dynamics, one molecule at a time

In a multicellular organism, each cell type expresses only a subset of genes encoded in the genome. Moreover, hundreds of genes can be regulated by external stimuli. This complex regulation is mediated by proteins called transcription factors. This special type of proteins can bind to specific sequences in the genome called response elements, within regulatory regions known as enhancers. Upon binding, transcription factors serve as a “scaffold” to recruit several other proteins such as cofactors and chromatin remodelers that would ultimately promote the up- (or down) regulation of genes.

Fig. 1. Single molecules of the glucocorticoid receptor in living cells. Each green dot represents a single molecule. The nucleus (in blue) is delimited by the white-dashed line. Scale bar 6 µm.

For decades, it was thought that transcription factors stay bound to chromatin for long periods of time, allowing the sequential and well-ordered recruitment of the entire transcriptional machine. However, with the advent of live-cell fluorescence microscopy imaging, it has been demonstrated that most of transcription factors interact very transiently with DNA, in the order of just a few seconds. This radical change in paradigm raised several questions about the dynamic nature of gene expression regulation, which is still largely unresolved.

New technological advances in organic fluorophore development, microscopy techniques, and camera sensitivity have allowed researchers to visualize individual protein molecules in live cells with an unprecedent spatial and temporal resolution. This allows us to directly quantify the time each individual transcription factor remain bound to chromatin. In our study, we describe the methodology used to visualize and track the dynamics of transcription factors at the single-molecule level (Single-molecule Tracking, SMT), and further discuss the current challenges this exciting new field faces.

To be able to visualize and follow individual fluorescent proteins, it is imperative to work with the brightest, more stable fluorophore available. Unfortunately, genetically encoded fluorescent proteins such as GFP are too dim, and bleach to rapidly, to be of use. A better alternative employs organic fluorophore dyes, including reagents developed by the Lavis Lab at Janelia Farms. These dyes can be irreversibly bound to small proteins with high affinity binding pockets (HaloTag, SNAP-tag and CLIP-tag). By making fusion chimeras between the tags and the protein of interest, it is possible to visualize a specific transcription factor with a bright and relatively stable fluorophore. Interestingly, we found that the same dye can have different photostability behavior depending on which tag protein they are attached to. Unfortunately, this raises some concerns regarding the possibility of combining multiple tags for multi-color applications.

From the optics perspective, to capture the light emanating from a single-molecule, and discriminate it from background, requires the reduction of out-of-focus illumination. This can be achieved using “highly inclined and laminated optical sheet illumination” (HiLO), developed by the Sakata-Sogawa lab. In this special imaging mode, a laser beam hits the sample from the side instead as from below, the most common configuration. This generates a thin, oblique layer of illumination across the cell nucleus. The caveat of this type of illumination is that photobleaching is not homogenous within cells, generating possible artifacts during the data analysis. The best way to analyze SMT data is a matter of continuous debate on this relatively new field. Nevertheless, all evidence derived from SMT thus far confirms the general idea that transcription factors are highly dynamic, and only a small fraction are specifically bound to response elements at any given time. Which of these molecules are functionally active, i.e. able to recruit RNA polymerase II, is still an open question that occupies many investigators currently working in this field.

Diego M. Presman, Gordon L. Hager
Laboratory of Receptor Biology and Gene Expression, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland, USA

Publication

Quantifying transcription factor binding dynamics at the single-molecule level in live cells.
Presman DM, Ball DA, Paakinaho V, Grimm JB, Lavis LD, Karpova TS, Hager GL
Methods. 2017 Jul 1

January 11, 2018