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Insect Biochemistry and Molecular... Jul 2023Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary...
Salivary glands are vital to tick feeding success and also play a crucial role in tick-borne pathogen transmission. In previous studies of Ixodes scapularis salivary glands, we demonstrated that saliva-producing type II and III acini are innervated by neuropeptidergic axons which release different classes of neuropeptides via their terminals (Šimo et al., 2009b, 2013). Among these, the neuropeptide SIFamide-along with its cognate receptor-were postulated to control the basally located acinar valve via basal epithelial and myoepithelial cells (Vancová et al., 2019). Here, we functionally characterized a second SIFamide receptor (SIFa_R2) from the I. scapularis genome and proved that it senses a low nanomolar level of its corresponding ligand. Insect SIFamide paralogs, SMYamides, also activated the receptor but less effectively compared to SIFamide. Bioinformatic and molecular dynamic analyses suggested that I. scapularis SIFamide receptors are class A GPCRs where the peptide amidated carboxy-terminus is oriented within the receptor binding cavity. The receptor was found to be expressed in Ixodes ricinus salivary glands, synganglia, midguts, trachea, and ovaries, but not in Malpighian tubules. Investigation of the temporal expression patterns suggests that the receptor transcript is highly expressed in unfed I. ricinus female salivary glands and then decreases during feeding. In synganglia, a significant transcript increase was detected in replete ticks. In salivary gland acini, an antibody targeting the SIFa_R2 recognized basal epithelial cells, myoepithelial cells, and basal granular cells in close proximity to the SIFamide-releasing axon terminals. Immunoreactivity was also detected in specific neurons distributed throughout various I. ricinus synganglion locations. The current findings, alongside previous reports from our group, indicate that the neuropeptide SIFamide acts via two different receptors that regulate distinct or common cell types in the basal region of type II and III acini in I. ricinus salivary glands. Our study investigates the peptidergic regulation of the I. ricinus salivary gland in detail, emphasizing the complexity of this system.
Topics: Female; Animals; Ixodes; Salivary Glands; Neurons; Saliva; Neuropeptides
PubMed: 37257628
DOI: 10.1016/j.ibmb.2023.103963 -
Basal and apical regulation of VEGF-A and placenta growth factor in the RPE/choroid and primary RPE.Molecular Vision 2015Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration...
PURPOSE
Members of the vascular endothelial growth factor (VEGF) family are strongly involved in pathological processes in the retina, such as age-related macular degeneration and diabetic retinopathy. Cells of the retinal pigment epithelium (RPE) constitutively secrete VEGF-A, and the secretion of placental growth factor (PlGF) has also been described. RPE cells are strongly polarized cells with different secretome at the apical and basal side. In this study, we evaluated the basal and apical regulation of VEGF-A and PlGF secretion in RPE/choroid explants and primary RPE cells.
METHODS
RPE/choroid tissue explants were prepared from porcine eyes and cultivated in modified Ussing chambers, separating apical (RPE) and basal (choroid) supernatant. Primary RPE cells were also prepared from porcine eyes and cultivated on Transwell plates. Explants and cells were treated with inhibitors for VEGFR-2 (SU1498), p38 (SB203580), and the transcription factors nuclear factor-kappa B (NF-κB) and SP-1 (mithramycin), respectively. VEGF-A and PlGF content was evaluated with enzyme-linked immunosorbent assay (ELISA). In addition, western blots were performed.
RESULTS
In the RPE/choroid, VEGF-A can initially be found on the apical and basal sides with significantly more pronounced secretion on the basal side. VEGF-A secretion is differentially regulated on the apical and basal sides, with the inhibition of SP-1 and NF-κB showing strong effects apically and basally after 24 h and 48 h, the inhibition of p38 displaying its effect mainly on the basal side with some effect apically after 48 h, and the inhibition of VEGFR-2 reducing the secretion of VEGF only on the apical side at 24 h and 48 h. In the RPE cell culture, similar effects were found, with inhibition of NF-κB or SP-1 displaying a strong decrease in VEGF-A on both sides, and p38 inhibition displaying only an inhibitory effect on the basal side. In contrast, an apical effect of VEGFR-2 inhibition was not found. However, the western blot experiments exhibited a significant decrease in the VEGF-A protein under SU1498 treatment. In the RPE/choroid organ cultures, PlGF was initially found mainly on the basal site with only minute amounts of PlGF found apically. NF-κB and SP-1 were strongly involved in PlGF regulation apically and basally, while VEGFR2 and to a lesser degree p38 displayed some regulation at the basal site. In the primary RPE cell culture, PlGF was not found on the apical or basal side.
CONCLUSIONS
VEGF-A and PlGF were constitutively secreted and regulated by the RPE/choroid complex, with PlGF secreted mainly by the choroid. Although the transcription factors NF-κB and SP-1 were involved in apical and basal regulation of both growth factors, VEGFR-2 displayed a strong polarity, with regulation of apical VEGF-A and basal PlGF secretion.
Topics: Animals; Cells, Cultured; Choroid; NF-kappa B; Organ Culture Techniques; Placenta Growth Factor; Pregnancy Proteins; Retinal Pigment Epithelium; Sp1 Transcription Factor; Sus scrofa; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; p38 Mitogen-Activated Protein Kinases
PubMed: 26167115
DOI: No ID Found -
Brain Research Feb 2008In this review we focus on the mechanisms, regulation, and cellular consequences of nuclear migration in the developing retina. In the nervous system, nuclear migration... (Review)
Review
In this review we focus on the mechanisms, regulation, and cellular consequences of nuclear migration in the developing retina. In the nervous system, nuclear migration is prominent during both proliferative and post-mitotic phases of development. Interkinetic nuclear migration is the process where the nucleus oscillates from the apical to basal surfaces in proliferative neuroepithelia. Proliferative nuclear movement occurs in step with the cell cycle, with M-phase being confined to the apical surface and G1-, S-, and G2-phases occurring at more basal locations. Later, following cell cycle exit, some neuron precursors migrate by nuclear translocation. In this mode of cellular migration, nuclear movement is the driving force for motility. Following discussion of the key components and important regulators for each of these processes, we present an emerging model where interkinetic nuclear migration functions to distinguish cell fates among retinal neuroepithelia.
Topics: Animals; Cell Cycle; Cell Lineage; Cell Movement; Cell Nucleus; Humans; Models, Biological; Neuroepithelial Cells; Neurons; Retina; Stem Cells
PubMed: 17560964
DOI: 10.1016/j.brainres.2007.05.021 -
Current Biology : CB May 2022As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the...
As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination, mechanical feedback, perhaps via adherens junctions, has been suggested to play a role. However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture. In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM), which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis. To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.
Topics: Cell Cycle; Cell Nucleus; Epithelium; Mechanotransduction, Cellular; Mitosis
PubMed: 35338851
DOI: 10.1016/j.cub.2022.03.004 -
Developmental Biology Jan 2022While the epithelial cell cortex displays profound asymmetries in protein distribution and morphology along the apico-basal axis, the extent to which the cytoplasm is...
While the epithelial cell cortex displays profound asymmetries in protein distribution and morphology along the apico-basal axis, the extent to which the cytoplasm is similarly polarized within epithelial cells remains relatively unexplored. We show that cytoplasmic organelles within C. elegans embryonic intestinal cells develop extensive apico-basal polarity at the time they establish cortical asymmetry. Nuclei and conventional endosomes, including early endosomes, late endosomes, and lysosomes, become polarized apically. Lysosome-related gut granules, yolk platelets, and lipid droplets become basally enriched. Removal of par-3 activity does not disrupt organelle positioning, indicating that cytoplasmic apico-basal asymmetry is independent of the PAR polarity pathway. Blocking the apical migration of nuclei leads to the apical positioning of gut granules and yolk platelets, whereas the asymmetric localization of conventional endosomes and lipid droplets is unaltered. This suggests that nuclear positioning organizes some, but not all, cytoplasmic asymmetries in this cell type. We show that gut granules become apically enriched when WHT-2 and WHT-7 function is disrupted, identifying a novel role for ABCG transporters in gut granule positioning during epithelial polarization. Analysis of WHT-2 and WHT-7 ATPase mutants is consistent with a WHT-2/WHT-7 heterodimer acting as a transporter in gut granule positioning. In wht-2(-) mutants, the polarized distribution of other organelles is not altered and gut granules do not take on characteristics of conventional endosomes that could have explained their apical mispositioning. During epithelial polarization wht-2(-) gut granules exhibit a loss of the Rab32/38 family member GLO-1 and ectopic expression of GLO-1 is sufficient to rescue the basal positioning of wht-2(-) and wht-7(-) gut granules. Furthermore, depletion of GLO-1 causes the mislocalization of the endolysosomal RAB-7 to gut granules and RAB-7 drives the apical mispositioning of gut granules when GLO-1, WHT-2, or WHT-7 function is disrupted. We suggest that ABC transporters residing on gut granules can regulate Rab dynamics to control organelle positioning during epithelial polarization.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Polarity; Epithelial Cells; Intestinal Mucosa; Organelles
PubMed: 34597675
DOI: 10.1016/j.ydbio.2021.09.007 -
Advanced Healthcare Materials Feb 2021This paper describes mammary organoids with a basal-in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials...
This paper describes mammary organoids with a basal-in phenotype where the basement membrane is located on the interior surface of the organoid. A key materials consideration to induce this basal-in phenotype is the use of a minimal gel scaffold that the epithelial cells self-assemble around and encapsulate. When MDA-MB-231 breast cancer cells are co-cultured with epithelial cells from day 0 under these conditions, cells self-organize into patterns with distinct cancer cell populations both inside and at the periphery of the epithelial organoid. In another type of experiment, the robust formation of the basement membrane on the epithelial organoid interior enables convenient studies of MDA-MB-231 invasion in a tumor progression-relevant direction relative to epithelial cell-basement membrane positioning. That is, the study of cancer invasion through the epithelium first, followed by the basement membrane to the basal side, is realized in an experimentally convenient manner where the cancer cells are simply seeded on the outside of preformed organoids, and their invasion into the organoid is monitored. Interestingly, invasion is more prominent when tumor cells are added to day 7 organoids with less developed basement membranes compared to day 16 organoids with more defined ones.
Topics: Basement Membrane; Epithelial Cells; Humans; Neoplasm Invasiveness; Organoids; Phenotype
PubMed: 32583612
DOI: 10.1002/adhm.202000810 -
Methods in Molecular Biology (Clifton,... 2020The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be... (Comparative Study)
Comparative Study
The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be expanded in cell culture and instructed to differentiate at an air-liquid interface using transwell membranes and differentiation media. For basal cell expansion, we have used 3T3-J2 co-culture in epithelial culture medium containing EGF, insulin, and a RHO-associated protein kinase (ROCK) inhibitor, Y-27632 (3T3 + Y). Here we describe our protocols for ciliated differentiation of these cultures at air-liquid interface and compare four commercially available differentiation media, across nine donor cell cultures (six healthy, two patients with chronic obstructive pulmonary disease (COPD), and one with primary ciliary dyskinesia (PCD)). Bright-field and immunofluorescence imaging suggested broad similarity between differentiation protocols. Subtle differences were seen in transepithelial electrical resistance (TEER), ciliary beat frequency, mucus production, and the extent to which basal cells are retained in differentiated cultures. Overall, the specific differentiation medium used in our air-liquid interface culture protocol was not a major determinant of ciliation, and our data suggest that the differentiation potential of basal cells at the outset is a more critical factor in air-liquid interface culture outcome. Detailed information on the constituents of the differentiation media was only available from one of the four manufacturers, a factor that may have profound implications in the interpretation of some research studies.
Topics: 3T3 Cells; Animals; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Coculture Techniques; Culture Media; Epithelial Cells; Humans; Mice; Nasal Mucosa; Stem Cells
PubMed: 31707647
DOI: 10.1007/7651_2019_269 -
International Journal of Molecular... Nov 2015The human interfollicular epidermis is renewed throughout life by populations of proliferating basal keratinocytes. Though interfollicular keratinocyte stem cells have... (Review)
Review
The human interfollicular epidermis is renewed throughout life by populations of proliferating basal keratinocytes. Though interfollicular keratinocyte stem cells have been identified, it is not known how self-renewal in this compartment is spatially organized. At the epidermal-dermal junction, keratinocytes sit atop a heterogeneous mix of dermal cells that may regulate keratinocyte self-renewal by influencing local tissue architecture and signalling microenvironments. Focusing on the rete ridges and complementary dermal papillae in human skin, we review the identity and organisation of abundant dermal cells types and present evidence for interactions between the dermal microenvironment and the interfollicular keratinocytes.
Topics: Adipocytes; Animals; Basement Membrane; Capillaries; Cell Differentiation; Cellular Microenvironment; Endothelial Cells; Epidermal Cells; Epidermis; Fibroblasts; Humans; Keratinocytes; Pericytes; Regeneration; Regenerative Medicine; Stem Cells
PubMed: 26602926
DOI: 10.3390/ijms161226078 -
PLoS Biology Sep 2022In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane....
In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.
Topics: Animals; Cell Membrane; Centrioles; Cilia; Mammals; Paramecium
PubMed: 36070319
DOI: 10.1371/journal.pbio.3001782 -
ELife Nov 2022Epithelial tissues acquire their integrity and function through the apico-basal polarization of their constituent cells. Proteins of the PAR and Crumbs complexes are...
Epithelial tissues acquire their integrity and function through the apico-basal polarization of their constituent cells. Proteins of the PAR and Crumbs complexes are pivotal to epithelial polarization, but the mechanistic understanding of polarization is challenging to reach, largely because numerous potential interactions between these proteins and others have been found, without a clear hierarchy in importance. We identify the regionalized and segregated organization of members of the PAR and Crumbs complexes at epithelial apical junctions by imaging endogenous proteins using stimulated-emission-depletion microscopy on Caco-2 cells, and human and murine intestinal samples. Proteins organize in submicrometric clusters, with PAR3 overlapping with the tight junction (TJ) while PALS1-PATJ and aPKC-PAR6β form segregated clusters that are apical of the TJ and present in an alternated pattern related to actin organization. CRB3A is also apical of the TJ and partially overlaps with other polarity proteins. Of the numerous potential interactions identified between polarity proteins, only PALS1-PATJ and aPKC-PAR6β are spatially relevant in the junctional area of mature epithelial cells, simplifying our view of how polarity proteins could cooperate to drive and maintain cell polarity.
Topics: Humans; Animals; Mice; Caco-2 Cells; Epithelium; Tight Junctions; Epithelial Cells; Microscopy
PubMed: 36341714
DOI: 10.7554/eLife.62087