scholarly journals A transgenic toolkit for visualizing and perturbing microtubules in mice reveals unexpected functions for non-centrosomal arrays in epidermal morphogenesis

2017 ◽  
Author(s):  
Andrew Muroyama ◽  
Terry Lechler
Keyword(s):  
Cell Types ◽  
Microtubule Dynamics ◽  
Microtubule Organization ◽  
Strong Suppression ◽  
Physiological Functions ◽  
Microtubule Growth ◽  
Epidermal Morphogenesis ◽  
Dynamics And Function ◽  
And Function

AbstractDifferentiation induces reorganization of microtubules (MTs) into non-centrosomal arrays in a variety of tissues. The physiological functions of these microtubule arrays are just beginning to be understood as few tools currently exist to genetically perturb microtubule organization in vivo, particularly in mammals. We developed a genetic toolkit that can be broadly applied to the study of microtubule dynamics and function in many cell types. Using a TRE-EB1-GFP mouse we demonstrate that distinct differentiation transitions in the epidermis cause a decrease in microtubule growth rates and microtubule growth lifetimes, resulting in strong suppression of dynamics. To understand the physiological functions of these stable, non-centrosomal microtubules, we generated a TRE-spastin mouse, which can be used to perturb microtubule organization in a wide-variety of tissues in vivo. Unexpectedly, microtubule perturbation exclusively in post-mitotic keratinocytes had profound consequences on epidermal morphogenesis. We uncoupled novel cell-autonomous roles for MTs in differentiation-driven cell flattening from non-cell autonomous functions in regulating proliferation, differentiation, and tissue architecture. Taken together, we have created tools that will be broadly useful for the study of microtubule dynamics and function in mammalian tissue physiology and have used them to uncover previously unknown functions for non-centrosomal microtubules during mammalian epidermal development.

scholarly journals A transgenic toolkit for visualizing and perturbing microtubules reveals unexpected functions in the epidermis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Andrew Muroyama ◽  
Terry Lechler
Keyword(s):  
Growth Rate ◽  
Cell Types ◽  
Specific Cell ◽  
Tissue Architecture ◽  
Physiological Functions ◽  
Epidermal Morphogenesis ◽  
Dynamics And Function ◽  
And Function ◽  
Genetic Toolkit ◽  
Epidermal Development

The physiological functions of microtubules (MTs) are poorly understood in many differentiated cell types. We developed a genetic toolkit to study MT dynamics and function in diverse cells. Using TRE-EB1-GFP mice, we found that MT dynamics are strongly suppressed in differentiated keratinocytes in two distinct steps due to alterations in both growth rate and lifetime. To understand the functions of these MT populations, we developed TRE-spastin mice to disrupt MTs in specific cell types. MT perturbation in post-mitotic keratinocytes had profound consequences on epidermal morphogenesis. We uncoupled cell-autonomous roles in cell flattening from non-cell-autonomous requirements for MTs in regulating proliferation, differentiation, and tissue architecture. This work uncovers physiological roles for MTs in epidermal development, and the tools described here will be broadly useful to study MT dynamics and functions in mammals.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

scholarly journals The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Thomas S McAlear ◽  
Susanne Bechstedt
Keyword(s):  
Cell Division ◽  
Growth Factor ◽  
Mitotic Spindle ◽  
Apparent Rate Constant ◽  
Microtubule Dynamics ◽  
Proliferation Marker ◽  
Microtubule Growth ◽  
Large Rearrangements ◽  
And Function

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

scholarly journals Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

2018 ◽  
Author(s):  
Caroline Fecher ◽  
Laura Trovò ◽  
Stephan A. Müller ◽  
Nicolas Snaidero ◽  
Jennifer Wettmarshausen ◽  
...  
Keyword(s):  
Molecular Diversity ◽  
Molecular Level ◽  
Cell Types ◽  
Long Chain Fatty Acids ◽  
Cell Type ◽  
Mitochondrial Proteome ◽  
Novel Approach ◽  
Cell Type Specific ◽  
And Function

AbstractMitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

scholarly journals Polymorphonuclear Leukocytes Prepared by Continuous-Flow Filtration Leukapheresis: Viability and Function

Blood ◽  
1974 ◽  
Vol 44 (5) ◽  
pp. 707-713 ◽  
Author(s):  
Michael B. Harris ◽  
Isaac Djerassi ◽  
Elias Schwartz ◽  
Richard K. Root
Keyword(s):  
Continuous Flow ◽  
Polymorphonuclear Leukocytes ◽  
Cell Types ◽  
High Yield ◽  
Trypan Blue Exclusion ◽  
Flow Filtration ◽  
And Function ◽  
Combating Infection

Abstract Preparation of granulocytes for transfusion in high yield and relatively free of contamination by other cell types has been made possible by the technique of continuous-flow filtration leukapheresis (CFFL). Since previous work suggested that granulocytes collected in this manner may have impaired viability and function, a detailed study of the bactericidal, metabolic, and chemotactic properties of such cells was performed and compared to control cells obtained from the same donors prior to CFFL. The granulocyte percentage of the cell suspensions obtained by CFFL averaged 94.5% ± 1.5% compared to 82.5% ± 1.8% for the controls (p < 0.001) with viability of the PMNs determined by trypan blue exclusion being 97.5% ± 0.9% and 98.2% ± 0.5%, respectively. The phogocytic, metabolic (14C-I-glucose oxidation and protein iodination) and chemotactic properties of both cell types were equivalent in suspensions equalized for granulocyte content. These findings indicate that CFFL technique employed does not impair granulocyte viability or function in vitro. Studies of the in vivo survival and function of CFFL granulocytes are necessary to evaluate their efficacy in combating infection in severely leukopenic patients.

P0658MATRIX AND FLOW MODULATE GENE EXPRESSION IN A 3D VASCULARISED TUBULE MODEL

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Julie Williams ◽  
Sanlin Robinson ◽  
Babak Alaei ◽  
Kimberly Homan ◽  
Maryam Clausen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Impact Analysis ◽  
Cell Types ◽  
Drug Uptake ◽  
Shear Stresses ◽  
The Matrix ◽  
And Function

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.

scholarly journals Dynamics and Function of Langerhans Cells In Vivo

Immunity ◽  
2005 ◽  
Vol 22 (5) ◽  
pp. 643-654 ◽  
Author(s):  
Adrien Kissenpfennig ◽  
Sandrine Henri ◽  
Bertrand Dubois ◽  
Corinne Laplace-Builhé ◽  
Pierre Perrin ◽  
...  
Keyword(s):  
Langerhans Cells ◽  
Dynamics And Function ◽  
And Function

scholarly journals The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease

2008 ◽  
Vol 205 (9) ◽  
pp. 2005-2017 ◽  
Author(s):  
Mark M.W. Chong ◽  
Jeffrey P. Rasmussen ◽  
Alexander Y. Rudensky ◽  
Dan R. Littman
Keyword(s):  
T Cell ◽  
Inflammatory Disease ◽  
Cell Types ◽  
Mirna Biogenesis ◽  
Cell Compartment ◽  
Foxp3 Gene ◽  
Genetic Ablation ◽  
And Function ◽  
Specific Deletion

MicroRNAs (miRNAs) are implicated in the differentiation and function of many cell types. We provide genetic and in vivo evidence that the two RNaseIII enzymes, Drosha and Dicer, do indeed function in the same pathway. These have previously been shown to mediate the stepwise maturation of miRNAs (Lee, Y., C. Ahn, J. Han, H. Choi, J. Kim, J. Yim, J. Lee, P. Provost, O. Radmark, S. Kim, and V.N. Kim. 2003. Nature. 425:415–419), and genetic ablation of either within the T cell compartment, or specifically within Foxp3+ regulatory T (T reg) cells, results in identical phenotypes. We found that miRNA biogenesis is indispensable for the function of T reg cells. Specific deletion of either Drosha or Dicer phenocopies mice lacking a functional Foxp3 gene or Foxp3+ cells, whereas deletion throughout the T cell compartment also results in spontaneous inflammatory disease, but later in life. Thus, miRNA-dependent regulation is critical for preventing spontaneous inflammation and autoimmunity.

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