Dsg2 ectodomain organization increases throughout desmosome assembly.

Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Fluidic shear stress alters clathrin dynamics and vesicle formation in endothelial cells.

Endothelial cells (ECs) experience a variety of highly dynamic mechanical stresses. Among others, cyclic stretch and increased plasma membrane tension inhibit clathrin-mediated endocytosis (CME) in non-ECs cells. How ECs overcome such unfavorable, from biophysical perspective, conditions and maintain CME remains elusive. Previously, we have used simultaneous two-wavelength axial ratiometry (STAR) microscopy to show that endocytic dynamics are similar between statically cultured human umbilical vein endothelial cells (HUVECs) and fibroblast-like Cos-7 cells. Here we asked whether biophysical stresses generated by blood flow could favor one mechanism of clathrin-coated vesicle formation to overcome environment present in vasculature. We used our data processing platform - DrSTAR - to examine if clathrin dynamics are altered in HUVECs grown under fluidic sheer stress (FSS). Surprisingly, we found that FSS led to an increase in clathrin dynamics. In HUVECs grown under FSS we observed a 2.3-fold increase in clathrin-coated vesicle formation and a 1.9-fold increase in non-productive flat clathrin lattices compared to cells grown in static conditions. The curvature-positive events had significantly delayed curvature initiation in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Overall, our findings indicate that clathrin dynamics and CCV formation can be modulated by the local physiological environment and represents an important regulatory mechanism.

Site-specific phosphorylations of the Arf activator GBF1 differentially regulate GBF1 function in Golgi homeostasis and secretion versus cytokinesis.

Diverse cellular processes, including membrane traffic, lipid homeostasis, cytokinesis, mitochondrial positioning, and cell motility are critically dependent on the Sec7 domain guanine nucleotide exchange factor GBF1. Yet, how the participation of GBF1 in a particular cellular function is regulated is unknown. Here, we show that the phosphorylation of specific highly conserved serine and tyrosine residues within the N-terminal domain of GBF1 differentially regulates its function in maintaining Golgi homeostasis and facilitating secretion versus its role in cytokinesis. Specifically, GBF1 mutants containing single amino acid substitutions that mimic a stably phosphorylated S233, S371, Y377, and Y515 or the S233A mutant that can't be phosphorylated are fully able to maintain Golgi architecture and support cargo traffic through the secretory pathway when assessed in multiple functional assays. However, the same mutants cause multi-nucleation when expressed in cells, and appear to inhibit the progression through mitosis and the resolution of cytokinetic bridges. Thus, GBF1 participates in distinct interactive networks when mediating Golgi homeostasis and secretion versus facilitating cytokinesis, and GBF1 integration into such networks is differentially regulated by the phosphorylation of specific GBF1 residues.

DrSTAR: Tracking real-time nanometer axial changes.

Protein interactions with the plasma membrane mediate processes critical for cell viability such as migration and endocytosis, yet our understanding of how recruitment of key proteins correlates with their ability to sense or induce energetically unfavorable plasma membrane shapes remains limited. Simultaneous two-wavelength axial ratiometry (STAR) microscopy provides millisecond time resolution and nanometer axial resolution of protein dynamics at the basal plasma membrane. However, STAR microscopy requires extensive and time-consuming quantitative data processing to access axial (Δz) information. Therefore, addressing questions about the influence of biological and biophysical factors on the interaction between the plasma membrane and protein of interest remains challenging. Here, we overcome the limitations in STAR data processing and present dynamic reference STAR (DrSTAR): a user-friendly, automated, open-source MATLAB-based package. DrSTAR enables processing multiple experimental conditions and biological replicates, employs a novel local background referencing algorithm, and accelerates processing time to facilitate broad adaptation of STAR for studying nanometer axial changes in protein distribution.

Imaging nanoscale axial dynamics at the basal plasma membrane.

Understanding of how energetically unfavorable plasma membrane shapes form, especially in the context of dynamic processes in living cells or tissues like clathrin-mediated endocytosis is in its infancy. Even though cutting-edge microscopy techniques that bridge this gap exist, they remain underused in biomedical sciences. Here, we demystify the perceived complexity of these advanced microscopy approaches and demonstrate their power in resolving nanometer axial dynamics in living cells. Total internal reflection fluorescence microscopy based approaches are the main focus of this review. We present clathrin-mediated endocytosis as a model system when describing the principles, data acquisition requirements, data interpretation strategies, and limitations of the described techniques. We hope this standardized description will bring the approaches for measuring nanoscale axial dynamics closer to the potential users and help in choosing the right approach to the right question.

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis.

Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.

Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics.

The establishment of apicobasal or planar cell polarity involves many events that occur at or near the plasma membrane including focal adhesion dynamics, endocytosis, exocytosis, and cytoskeletal reorganization. It is desirable to visualize these events without interference from other regions deeper within the cell. Total internal reflection fluorescence (TIRF) microscopy utilizes an elegant optical sectioning approach to visualize fluorophores near the sample-coverslip interface. TIRF provides high-contrast fluorescence images with limited background and virtually no out-of-focus light, ideal for visualizing and tracking dynamics near the plasma membrane. In this chapter, we present a general experimental and analysis TIRF pipeline for studying cell surface receptor endocytosis. The approach presented can be easily applied to study other dynamic biological processes at or near the plasma membrane using TIRF microscopy.

Biological aspects of the tongue morphology of wild-captive WWCPS rats: a histological, histochemical and ultrastructural study.

The aim of this study was to characterise the tongue in wild-type rats using several microscopic techniques. Warsaw Wild Captive Pisula Stryjek (WWCPS) rats belong to a lineage of wild-caught rats. The study was carried out on tongues of 15 male and 15 female WWCPS rats. Histological, histochemical and ultrastructural studies were carried out. There were no significant differences between the male and female WWCPS rat tongues. There was a median groove approximately 1 cm long in the apex of the tongue that faded caudally. The intermolar prominence was clearly marked in the distal part of the lingual body. Lingual mechanical papillae located on the surface of the tongue formed four subtypes based on their shape: small filiform papillae, giant filiform papillae, thin elongated filiform papillae and wide filiform papillae. Gustatory papillae formed the second group of papillae and were divided into bud-shaped fungiform papillae, a single vallate papilla surrounded by an incomplete papillary groove and foliate papillae, which were a well-formed and composed of several pairs of folds divided by longitudinal grooves. In the posterior lingual glands (mucoserous and serous), acidic sulphated mucin-secreting cells gave a strong AB pH 2.5 positive reaction, and a positive reaction with the AB pH 1.0 stain for acidic carboxylated mucin. Double AB/PAS staining showed the presence of the majority of mucous cells with predominant of acidic mucins. Positive PAS staining showed the presence of neutral mucin. HDI staining demonstrated a weak positive reaction within Weber's glands of the WWCPS rat tongue.

αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS.