isolation-identification-vascular-endothelial-cells-from-distinct

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

University of Delaware

Congenital aniridia is caused by heterozygous mutations in the PAX6 gene. In this disease, congenital iris and foveal hypoplasia is associated with juvenile onset cataract, glaucoma, and corneal keratopathy. In rodents, Pax6 mutations result in a congenital reduction in ocular size that is not typically described in human aniridia. Here, the ocular morphometry of aniridia patients is compared with the lens phenotype of Pax6 mice to reveal whether there are species differences in Pax6 regulation of lens development and homeostasis. Ultrasound biometry (UBM) revealed that eleven percent of aniridia patients exhibited mild microphthalmia while the anterior chamber depth of aniridic eyes was significantly reduced from 6 months of age onward. Although aniridic lens thickness was normal from birth, it was significantly decreased in aniridic lenses older than 30. Notably, 86% of aniridic lenses exhibited cataractous changes in this cohort. In addition, a significant proportion of aniridia patients develop lens subluxation as they age associated with reduced lens diameter as measured by anterior segment optical coherence tomography (AS-OCT). Analysis of young adult Pax6 mouse lenses by micro-computed tomography (microCT), bright field and dark field imaging revealed that they are reduced in size but did not exhibit overt cataracts at this age. Overall, this study reveals that congenital microphthalmia as assessed by axial length, or microphakia, as assessed by lens thickness, are not typical in human aniridia, although these are primary manifestations of Pax6 mutations in mice, suggesting that PAX6 regulates some aspects of lens development differently between these species.

Morphometric analysis of the lens in human aniridia and mouse Small eye.

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism.

The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Article	Year
Morphometric analysis of the lens in human aniridia and mouse Small eye. Experimental eye research\| PubMed ID: 33248069	2021
The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover. The Journal of biological chemistry\| PubMed ID: 34280433	2021
Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 35195928	2022

Richard West

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