scholarly journals Desmoplakin assembly dynamics in four dimensions

2005 ◽  
Vol 171 (6) ◽  
pp. 1045-1059 ◽  
Author(s):  
Lisa M. Godsel ◽  
Sherry N. Hsieh ◽  
Evangeline V. Amargo ◽  
Amanda E. Bass ◽  
Lauren T. Pascoe-McGillicuddy ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Particle Trajectory ◽  
De Novo ◽  
Time Lapse ◽  
Cell Contact ◽  
Four Dimensions ◽  
Tissue Integrity ◽  
Plakophilin 2 ◽  
Time Lapse Imaging ◽  
Cell Cell

The intermediate filament (IF)–binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell–cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 μm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell–cell contact and regulated by actin and DP–IF interactions.

scholarly journals Automated profiling of individual cell–cell interactions from high-throughput time-lapse imaging microscopy in nanowell grids (TIMING)

2015 ◽  
Vol 31 (19) ◽  
pp. 3189-3197 ◽  
Author(s):  
Amine Merouane ◽  
Nicolas Rey-Villamizar ◽  
Yanbin Lu ◽  
Ivan Liadi ◽  
Gabrielle Romain ◽  
...  
Keyword(s):  
High Throughput ◽  
Individual Cell ◽  
Time Lapse ◽  
Cell Interactions ◽  
Throughput Time ◽  
Time Lapse Imaging ◽  
Cell Cell

scholarly journals An Autism-Associated de novo Mutation in GluN2B Destabilizes Growing Dendrites by Promoting Retraction and Pruning

2021 ◽  
Vol 15 ◽  
Author(s):  
Jacob A. Bahry ◽  
Karlie N. Fedder-Semmes ◽  
Michael P. Sceniak ◽  
Shasta L. Sabo
Keyword(s):  
De Novo ◽  
Time Lapse ◽  
Autism Spectrum ◽  
Dendrite Growth ◽  
De Novo Mutation ◽  
Neocortical Neurons ◽  
Spectrum Disorders ◽  
Dendritic Branches ◽  
Dendrite Morphogenesis ◽  
Time Lapse Imaging

Mutations in GRIN2B, which encodes the GluN2B subunit of NMDA receptors, lead to autism spectrum disorders (ASD), but the pathophysiological mechanisms remain unclear. Recently, we showed that a GluN2B variant that is associated with severe ASD (GluN2B724t) impairs dendrite morphogenesis. To determine which aspects of dendrite growth are affected by GluN2B724t, we investigated the dynamics of dendrite growth and branching in rat neocortical neurons using time-lapse imaging. GluN2B724t expression shifted branch motility toward retraction and away from extension. GluN2B724t and wild-type neurons formed new branches at similar rates, but mutant neurons exhibited increased pruning of dendritic branches. The observed changes in dynamics resulted in nearly complete elimination of the net expansion of arbor size and complexity that is normally observed during this developmental period. These data demonstrate that ASD-associated mutant GluN2B interferes with dendrite morphogenesis by reducing rates of outgrowth while promoting retraction and subsequent pruning. Because mutant dendrites remain motile and capable of growth, it is possible that reducing pruning or promoting dendrite stabilization could overcome dendrite arbor defects associated with GRIN2B mutations.

scholarly journals Transcriptomic Coupling of PKP2 With Inflammatory and Immune Pathways Endogenous to Adult Cardiac Myocytes

2021 ◽  
Vol 11 ◽  
Author(s):  
Marta Pérez-Hernández ◽  
Grecia M. Marrón-Liñares ◽  
Florencia Schlamp ◽  
Adriana Heguy ◽  
Chantal J. M. van Opbergen ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Arrhythmogenic Right Ventricular Cardiomyopathy ◽  
Transcript Abundance ◽  
Cell Contact ◽  
Plakophilin 2 ◽  
Intracellular Signals ◽  
Adult Cardiac Myocytes ◽  
Inflammatory Immune Response ◽  
Cell Cell

Plakophilin-2 (PKP2) is classically defined as a component of the desmosome. Besides its role in cell–cell adhesion, PKP2 can modulate transcription through intracellular signals initiated at the site of cell–cell contact. Mutations in PKP2 associate with arrhythmogenic right ventricular cardiomyopathy (ARVC). Recent data demonstrate that inflammation plays a key role in disease progression; other results show an abundance of anti-heart antibodies in patients with confirmed diagnosis of ARVC. Here, we test the hypothesis that, in adult cardiac myocytes, PKP2 transcript abundance is endogenously linked to the abundance of transcripts participating in the inflammatory/immune response. Cardiac-specific, tamoxifen (TAM)-activated PKP2-knockout mice (PKP2cKO) were crossed with a RiboTag line to allow characterization of the ribosome-resident transcriptome of cardiomyocytes after PKP2 knockdown. Data were combined with informatics analysis of human cardiac transcriptome using GTEx. Separately, the presence of non-myocyte cells at the time of analysis was assessed by imaging methods. We identified a large number of transcripts upregulated consequent to PKP2 deficiency in myocytes, inversely correlated with PKP2 abundance in human transcriptomes, and part of functional pathways associated with inflammatory/immune responses. Our data support the concept that PKP2 is transcriptionally linked, in cardiac myocytes, to genes coding for host-response molecules even in the absence of exogenous triggers. Targeted anti-inflammatory therapy may be effective in ARVC.

scholarly journals SUMOylation of periplakin is critical for efficient reorganization of keratin filament network

2019 ◽  
Vol 30 (3) ◽  
pp. 357-369 ◽  
Author(s):  
Mansi Gujrati ◽  
Rohit Mittal ◽  
Lakhan Ekal ◽  
Ram Kumar Mishra
Keyword(s):  
Intermediate Filaments ◽  
Okadaic Acid ◽  
Intermediate Filament ◽  
Time Lapse ◽  
Intermediate Filament Proteins ◽  
Enhanced Sensitivity ◽  
Response To Stress ◽  
Keratin Filament ◽  
Time Lapse Imaging ◽  
Linker Domain

The architecture of the cytoskeleton and its remodeling are tightly regulated by dynamic reorganization of keratin-rich intermediate filaments. Plakin family proteins associate with the network of intermediate filaments (IFs) and affect its reorganization during migration, differentiation, and response to stress. The smallest plakin, periplakin (PPL), interacts specifically with intermediate filament proteins K8, K18, and vimentin via its C-terminal linker domain. Here, we show that periplakin is SUMOylated at a conserved lysine in its linker domain (K1646) preferentially by small ubiquitin-like modifier 1 (SUMO1). Our data indicate that PPL SUMOylation is essential for the proper reorganization of the keratin IF network. Stresses perturbing intermediate-filament and cytoskeletal architecture induce hyper-­SUMOylation of periplakin. Okadaic acid induced hyperphosphorylation-dependent collapse of the keratin IF network results in a similar hyper-SUMOylation of PPL. Strikingly, exogenous overexpression of a non-SUMOylatable periplakin mutant (K1646R) induced aberrant bundling and loose network interconnections of the keratin filaments. Time-lapse imaging of cells expressing the K1646R mutant showed the enhanced sensitivity of keratin filament collapse upon okadaic acid treatment. Our data identify an important regulatory role for periplakin SUMOylation in dynamic reorganization and stability of keratin IFs.

scholarly journals E-cadherin expression pattern during zebrafish embryonic epidermis development

F1000Research ◽  
2019 ◽  
Vol 7 ◽  
pp. 1489 ◽  
Author(s):  
María Florencia Sampedro ◽  
María Fernanda Izaguirre ◽  
Valeria Sigot
Keyword(s):  
Cell Shape ◽  
Basal Layer ◽  
Temporal Distribution ◽  
Cell Contact ◽  
3D Segmentation ◽  
Adhesion Receptor ◽  
Hexagonal Packing ◽  
Tissue Integrity ◽  
E Cadherin ◽  
Cell Cell

Background: E-cadherin is the major adhesion receptor in epithelial adherens junctions (AJs). On established epidermis, E-cadherin performs fine-tuned cell-cell contact remodeling to maintain tissue integrity, which is characterized by modulation of cell shape, size and packing density. In zebrafish, the organization and distribution of E-cadherin in AJs during embryonic epidermis development remain scarcely described. Methods: Combining classical immunofluorescence, deconvolution microscopy and 3D-segmentation of AJs in epithelial cells, a quantitative approach was implemented to assess the spatial and temporal distribution of E-cadherin across zebrafish epidermis between 24 and 72 hpf. Results: increasing levels of E-cadh protein parallel higher cell density and the appearance of hexagonal cells in the enveloping layer (EVL) as well as the establishments of new cell-cell contacts in the epidermal basal layer (EBL), being significantly between 31 and 48 hpf. Conclusions: Increasing levels of E-cadherin in AJs correlates with extensive changes in cell morphology towards hexagonal packing during the epidermis morphogenesis.

scholarly journals Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.

1996 ◽  
Vol 135 (6) ◽  
pp. 1899-1911 ◽  
Author(s):  
C L Adams ◽  
W J Nelson ◽  
S J Smith
Keyword(s):  
Cell Adhesion ◽  
Time Lapse ◽  
Cell Contact ◽  
Beta Catenin ◽  
Actin Reorganization ◽  
Cell Contacts ◽  
Spatial Ordering ◽  
Triton X ◽  
E Cadherin ◽  
Cell Cell

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.

scholarly journals Dynamic Migration and Cell-Cell Interactions of Early Reprogramming Revealed by High-Resolution Time-Lapse Imaging

Stem Cells ◽  
2013 ◽  
Vol 31 (5) ◽  
pp. 895-905 ◽  
Author(s):  
Cynthia M. Megyola ◽  
Yuan Gao ◽  
Alexandra M. Teixeira ◽  
Jijun Cheng ◽  
Kartoosh Heydari ◽  
...  
Keyword(s):  
High Resolution ◽  
Time Lapse ◽  
Cell Interactions ◽  
Resolution Time ◽  
Time Lapse Imaging ◽  
Cell Cell

scholarly journals Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

2015 ◽  
Vol 209 (3) ◽  
pp. 367-376 ◽  
Author(s):  
Katharina Grikscheit ◽  
Tanja Frank ◽  
Ying Wang ◽  
Robert Grosse
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Actin Polymerization ◽  
De Novo ◽  
Early Stage ◽  
Adhesion Formation ◽  
Actin Dynamics ◽  
Cell Contact ◽  
Actin Assembly ◽  
Cell Cell

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.

scholarly journals Integration of vascular progenitors into functional blood vessels represents a novel mechanism of vascular growth

2021 ◽  
Author(s):  
Sanjeeva Metikala ◽  
Michael Warkala ◽  
Satish Casie Chetty ◽  
Brendan Chestnut ◽  
Elizabeth Plender ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Blood Circulation ◽  
De Novo ◽  
Vascular Endothelial Cells ◽  
Time Lapse ◽  
Vascular Endothelial ◽  
Rna Seq ◽  
Cardinal Vein ◽  
Vascular Growth ◽  
Time Lapse Imaging

During embryogenesis, the initial vascular network is thought to form by the process of vasculogenesis, or the specification of vascular progenitors de novo. After the initial blood circulation has been established, the majority of later-forming vessels are thought to arise by angiogenesis from the already established vasculature. Here we show that new vascular progenitors in zebrafish embryos contribute to functional vasculature even after blood circulation has been established. Based on the expression analysis of early vascular progenitor markers etv2 and tal1, we characterized a novel site of late vasculogenesis (termed secondary vascular field, SVF), located bilaterally along the yolk extension. Using time-lapse imaging of etv2 reporter lines, we show that SVF cells migrate and incorporate into functional blood vessels and contribute to the formation of the posterior cardinal vein and subintestinal vasculature, suggesting a novel mode of vascular growth. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of etv2 function prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. In addition, we performed single-cell RNA-seq analysis to identify the transcriptional profile of SVF cells, which demonstrated similarities and differences between the transcriptomes of SVF cells and early vascular progenitors. Our results characterize a novel mechanism of how new vascular progenitors incorporate into established vasculature and revise our understanding of basic mechanisms that regulate vascular development.

scholarly journals Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation

2014 ◽  
Vol 25 (23) ◽  
pp. 3749-3764 ◽  
Author(s):  
Viktor Todorovic´ ◽  
Jennifer L. Koetsier ◽  
Lisa M. Godsel ◽  
Kathleen J. Green
Keyword(s):  
Initial Phase ◽  
Adherens Junction ◽  
Cell Contact ◽  
Functional Association ◽  
Plakophilin 2 ◽  
Plakophilin 3 ◽  
E Cadherin ◽  
Cell Cell

The pathways driving desmosome and adherens junction assembly are temporally and spatially coordinated, but how they are functionally coupled is poorly understood. Here we show that the Armadillo protein plakophilin 3 (Pkp3) mediates both desmosome assembly and E-cadherin maturation through Rap1 GTPase, thus functioning in a manner distinct from the closely related plakophilin 2 (Pkp2). Whereas Pkp2 and Pkp3 share the ability to mediate the initial phase of desmoplakin (DP) accumulation at sites of cell–cell contact, they play distinct roles in later steps: Pkp3 is required for assembly of a cytoplasmic population of DP-enriched junction precursors, whereas Pkp2 is required for transfer of the precursors to the membrane. Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly. We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing. These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

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