OOPS: Object-Oriented Polarization Software for analysis of fluorescence polarization microscopy images.

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.

Disruption of the creb3l1 gene causes defects in caudal fin regeneration and patterning in zebrafish Danio rerio.

BACKGROUND: The gene cAMP-Responsive Element Binding protein 3-like-1 (CREB3L1) has been implicated in bone development in mice, with CREB3L1 knock-out mice exhibiting fragile bones, and in humans, with CREB3L1 mutations linked to osteogenesis imperfecta. However, the mechanism through which Creb3l1 regulates bone development is not fully understood. RESULTS: To probe the role of Creb3l1 in organismal physiology, we used CRISPR-Cas9 genome editing to generate a Danio rerio (zebrafish) model of Creb3l1 deficiency. In contrast to mammalian phenotypes, the Creb3l1 deficient fish do not display abnormalities in osteogenesis, except for a decrease in the bifurcation pattern of caudal fin. Both, skeletal morphology and overall bone density appear normal in the mutant fish. However, the regeneration of caudal fin postamputation is significantly affected, with decreased overall regenerate and mineralized bone area. Moreover, the mutant fish exhibit a severe patterning defect during regeneration, with a significant decrease in bifurcation complexity of the fin rays and distalization of the bifurcation sites. Analysis of genes implicated in bone development showed aberrant patterning of shha and ptch2 in Creb3l1 deficient fish, linking Creb3l1 with Sonic Hedgehog signaling during fin regeneration. CONCLUSIONS: Our results uncover a novel role for Creb3l1 in regulating tissue growth and patterning during regeneration.

Lack of Nuclear Localization of the Creb3l1 Transcription Factor Causes Defects in Caudal Fin Bifurcation in Zebrafish Danio rerio.

INTRODUCTION: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. METHODS: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription. RESULTS: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. CONCLUSION: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

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. It remains elusive how ECs maintain CME in these biophysically unfavorable conditions. 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 influence CME. We used our data processing platform-DrSTAR-to examine if clathrin dynamics are altered in HUVECs after experiencing fluidic shear stress (FSS). We found that HUVECs cultivated under a physiological level of FSS had increased clathrin dynamics compared with static controls. FSS increased both clathrin-coated vesicle formation and nonproductive flat clathrin lattices by 2.3-fold and 1.9-fold, respectively. The curvature-positive events had significantly delayed curvature initiation relative to clathrin recruitment in flow-stimulated cells, highlighting a shift toward flat-to-curved clathrin transitions in vesicle formation. Overall, our findings indicate that clathrin dynamics and clathrin-coated vesicle formation can be modulated by the local physiological environment and represent an important regulatory mechanism.

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.