scholarly journals LIM kinase regulation of cytoskeletal dynamics is required for salivary gland branching morphogenesis

2014 ◽  
Vol 25 (16) ◽  
pp. 2393-2407 ◽  
Author(s):  
Shayoni Ray ◽  
Joseph A. Fanti ◽  
Diego P. Macedo ◽  
Melinda Larsen
Keyword(s):  
Salivary Gland ◽  
Late Stage ◽  
Ex Vivo ◽  
Branching Morphogenesis ◽  
Microtubule Assembly ◽  
Microtubule Organization ◽  
Lim Kinase ◽  
Cytoskeletal Dynamics ◽  
Assembly Factor ◽  
Focal Adhesion Protein

Coordinated actin microfilament and microtubule dynamics is required for salivary gland development, although the mechanisms by which they contribute to branching morphogenesis are not defined. Because LIM kinase (LIMK) regulates both actin and microtubule organization, we investigated the role of LIMK signaling in mouse embryonic submandibular salivary glands using ex vivo organ cultures. Both LIMK 1 and 2 were necessary for branching morphogenesis and functioned to promote epithelial early- and late-stage cleft progression through regulation of both microfilaments and microtubules. LIMK-dependent regulation of these cytoskeletal systems was required to control focal adhesion protein–dependent fibronectin assembly and integrin β1 activation, involving the LIMK effectors cofilin and TPPP/p25, for assembly of the actin- and tubulin-based cytoskeletal systems, respectively. We demonstrate that LIMK regulates the early stages of cleft formation—cleft initiation, stabilization, and progression—via establishment of actin stability. Further, we reveal a novel role for the microtubule assembly factor p25 in regulating stabilization and elongation of late-stage progressing clefts. This study demonstrates the existence of multiple actin- and microtubule-dependent stabilization steps that are controlled by LIMK and are required in cleft progression during branching morphogenesis.

scholarly journals Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury

2021 ◽  
Vol 22 (6) ◽  
pp. 3162
Author(s):  
Erni Sulistiyani ◽  
James M. Brimson ◽  
Ajjima Chansaenroj ◽  
Ladawan Sariya ◽  
Ganokon Urkasemsin ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Ex Vivo ◽  
Biological Effects ◽  
Branching Morphogenesis ◽  
Epithelial Proliferation ◽  
Expression Arrays ◽  
Gene Expression Arrays ◽  
Antioxidant Agents ◽  
Bright Field Microscopy ◽  
Injury Models

Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5–30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.

scholarly journals Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

2011 ◽  
Vol 192 (2) ◽  
pp. 321-334 ◽  
Author(s):  
Kenneth A. Myers ◽  
Kathryn T. Applegate ◽  
Gaudenz Danuser ◽  
Robert S. Fischer ◽  
Clare M. Waterman
Keyword(s):  
Endothelial Cell ◽  
Myosin Ii ◽  
Three Dimensional ◽  
Branching Morphogenesis ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Growth Speed ◽  
Two Dimensional ◽  
Cytoskeletal Dynamics ◽  
Microtubule Growth

During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end–tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II–mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs.

scholarly journals Annexin A2 Phosphorylation Mediates Cell Scattering and Branching Morphogenesis via Cofilin Activation

2007 ◽  
Vol 28 (3) ◽  
pp. 1029-1040 ◽  
Author(s):  
Marjo de Graauw ◽  
Ine Tijdens ◽  
Mirjam B. Smeets ◽  
Paul J. Hensbergen ◽  
André M. Deelder ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Three Dimensional ◽  
Branching Morphogenesis ◽  
Annexin A2 ◽  
Actin Binding ◽  
Lim Kinase ◽  
Cell Scattering ◽  
Cytoskeletal Dynamics ◽  
Upstream Kinase ◽  
And Migration

ABSTRACT Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.

scholarly journals In vitro models of fetal lung development to enhance research into congenital lung diseases

2021 ◽  
Author(s):  
Soichi Shibuya ◽  
Jessica Allen-Hyttinen ◽  
Paolo De Coppi ◽  
Federica Michielin
Keyword(s):  
Lung Development ◽  
Lung Diseases ◽  
Ex Vivo ◽  
Fetal Lung ◽  
Branching Morphogenesis ◽  
In Vitro Models ◽  
Normal Lung ◽  
Novel Therapeutic Strategies ◽  
Pseudoglandular Stage

Abstract Purpose This paper aims to build upon previous work to definitively establish in vitro models of murine pseudoglandular stage lung development. These can be easily translated to human fetal lung samples to allow the investigation of lung development in physiologic and pathologic conditions. Methods Lungs were harvested from mouse embryos at E12.5 and cultured in three different settings, i.e., whole lung culture, mesenchyme-free epithelium culture, and organoid culture. For the whole lung culture, extracted lungs were embedded in Matrigel and incubated on permeable filters. Separately, distal epithelial tips were isolated by firstly removing mesothelial and mesenchymal cells, and then severing the tips from the airway tubes. These were then cultured either in branch-promoting or self-renewing conditions. Results Cultured whole lungs underwent branching morphogenesis similarly to native lungs. Real-time qPCR analysis demonstrated expression of key genes essential for lung bud formation. The culture condition for epithelial tips was optimized by testing different concentrations of FGF10 and CHIR99021 and evaluating branching formation. The epithelial rudiments in self-renewing conditions formed spherical 3D structures with homogeneous Sox9 expression. Conclusion We report efficient protocols for ex vivo culture systems of pseudoglandular stage mouse embryonic lungs. These models can be applied to human samples and could be useful to paediatric surgeons to investigate normal lung development, understand the pathogenesis of congenital lung diseases, and explore novel therapeutic strategies.

Mesenchymal control over elongating and branching morphogenesis in salivary gland development

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 209-221
Author(s):  
Hiroyuki Nogawa ◽  
Takeo Mizuno
Keyword(s):  
Salivary Gland ◽  
Salivary Glands ◽  
Submaxillary Gland ◽  
Branching Morphogenesis ◽  
Mouse Submaxillary Gland ◽  
Gland Development

Recombination of the epithelium and mesenchyme between quail anterior submaxillary gland (elongating type) and quail anterior lingual or mouse submaxillary gland (branching type) was effected in vitro to clarify whether the elongating morphogenesis was directed by the epithelial or the mesenchymal component. Quail anterior submaxillary epithelium recombined with quail anterior lingual or mouse submaxillary mesenchyme came to branch. Conversely, quail anterior lingual or 12-day mouse submaxillary epithelium recombined with quail anterior submaxillary mesenchyme came to elongate, though the mesenchyme was less effective with 13-day mouse submaxillary epithelium. These results suggest that the elongating or branching morphogenesis of quail salivary glands is controlled by the mesenchyme.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

scholarly journals Structural insight into TPX2-stimulated microtubule assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Rui Zhang ◽  
Johanna Roostalu ◽  
Thomas Surrey ◽  
Eva Nogales
Keyword(s):  
Molecular Mechanism ◽  
Structural Information ◽  
Microtubule Assembly ◽  
Cryo Electron Microscopy ◽  
Interaction Mode ◽  
In Vitro Reconstitution ◽  
Assembly Factor ◽  
Ran Gtp ◽  
Mitosis And Meiosis

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.

scholarly journals Murine Cytomegalovirus Spread Depends on the Infected Myeloid Cell Type

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Helen E. Farrell ◽  
Kimberley Bruce ◽  
Clara Lawler ◽  
Philip G. Stevenson
Keyword(s):  
Dendritic Cells ◽  
Salivary Gland ◽  
Salivary Glands ◽  
Olfactory Epithelium ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Murine Cytomegalovirus ◽  
Cell Type ◽  
High Endothelial Venules ◽  
Brown Adipose

ABSTRACTCytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c+cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, and then entered the salivary glands, driven by constitutive signaling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal infection also delivered MCMV to the salivary glands via DC. However, it also seeded F4/80+infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infectedex vivothen adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80+cells in BAT and CD11c+cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to pertussis toxin inhibition of the GPCR signaling through Gi/osubstrate, whereas salivary gland colonization was sensitive. Since salivary gland infection required both M33 and Gi/o-coupled signaling, whereas BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections.IMPORTANCECytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With murine CMV (MCMV) we can track infected myeloid cells and so understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands. Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the “wrong” monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes.

scholarly journals Similar ex vivo expansion and post-irradiation regenerative potential of juvenile and aged salivary gland stem cells

2015 ◽  
Vol 116 (3) ◽  
pp. 443-448 ◽  
Author(s):  
Martti Maimets ◽  
Reinier Bron ◽  
Gerald de Haan ◽  
Ronald van Os ◽  
Robert P. Coppes
Keyword(s):  
Stem Cells ◽  
Salivary Gland ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Post Irradiation

scholarly journals Ror2 regulates branching, differentiation, and actin-cytoskeletal dynamics within the mammary epithelium

2015 ◽  
Vol 208 (3) ◽  
pp. 351-366 ◽  
Author(s):  
Kevin Roarty ◽  
Amy N. Shore ◽  
Chad J. Creighton ◽  
Jeffrey M. Rosen
Keyword(s):  
Wnt Signaling ◽  
Mammary Epithelium ◽  
Branching Morphogenesis ◽  
Mammary Development ◽  
Mammary Epithelial ◽  
Gene Level ◽  
Cytoskeletal Dynamics ◽  
Morphogenesis In Vitro

Wnt signaling encompasses β-catenin–dependent and –independent networks. How receptor context provides Wnt specificity in vivo to assimilate multiple concurrent Wnt inputs throughout development remains unclear. Here, we identified a refined expression pattern of Wnt/receptor combinations associated with the Wnt/β-catenin–independent pathway in mammary epithelial subpopulations. Moreover, we elucidated the function of the alternative Wnt receptor Ror2 in mammary development and provided evidence for coordination of this pathway with Wnt/β-catenin–dependent signaling in the mammary epithelium. Lentiviral short hairpin RNA (shRNA)-mediated depletion of Ror2 in vivo increased branching and altered the differentiation of the mammary epithelium. Microarray analyses identified distinct gene level alterations within the epithelial compartments in the absence of Ror2, with marked changes observed in genes associated with the actin cytoskeleton. Modeling of branching morphogenesis in vitro defined specific defects in cytoskeletal dynamics accompanied by Rho pathway alterations downstream of Ror2 loss. The current study presents a model of Wnt signaling coordination in vivo and assigns an important role for Ror2 in mammary development.

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