scholarly journals SPECC1L-deficient palate mesenchyme cells show speed and directionality defect

2019 ◽  
Author(s):  
Jeremy P. Goering ◽  
Dona Greta Isai ◽  
Everett G. Hall ◽  
Nathan R. Wilson ◽  
Edina Kosa ◽  
...  
Keyword(s):  
Wound Repair ◽  
Cell Tracking ◽  
Time Lapse ◽  
Primary Role ◽  
Palatal Shelf ◽  
Collective Movement ◽  
Primary Mouse ◽  
Mesenchyme Cells ◽  
Delayed Closure

AbstractClefts of the lip and/or palate (CL/P) are common anomalies that occur in 1/800 live births. Pathogenic SPECC1L variants identified in patients with rare atypical clefts and syndromic CL/P suggest the gene plays a primary role in face and palate development. We have generated Specc1l gene-trap (Specc1lcGT) and truncation (Specc1lΔC510) alleles that cause embryonic or perinatal lethality, respectively. Specc1lcGT/ΔC510 compound mutants show delayed and abnormal palatal shelf elevation at E14.5. By E15.5, the mutant shelves do elevate and fuse, however, the palatal rugae form abnormally. Palatogenesis requires extensive mesenchymal remodeling, especially during palatal shelf elevation. We posit that this remodeling involves collective movement of neural crest-derived palatal mesenchyme cells. Live time-lapse microscopy was performed to visualize in vitro wound-repair assays with wildtype and SPECC1L-deficient primary mouse embryonic palatal mesenchyme (MEPM) cells. SPECC1L-deficient MEPM cells consistently showed delayed closure in wound-repair assays. To evaluate which features of cellular movement were responsible, we performed automated particle image velocimetry (PIV) and manual cell tracking. The analyses revealed that both cell speed and directionality are disrupted in SPECC1L-deficient cells compared to controls. To determine if primary MEPM cells can move collectively, we assayed stream formation, which is a hallmark of collective movement. Indeed, MEPM cultures displayed correlated movement of neighboring cells. Importantly, correlation length was reduced in SPECC1L-deficient cultures, consistent with a role for SPECC1L in collective migration. Furthermore, we demonstrated that activation of the PI3K-AKT pathway with the 740Y-P small molecule can rescue the wound-closure delay in SPECC1L-deficient MEPM cells. Cell tracking analyses showed that this rescue was due to both increased speed and improved directionality. Altogether, our data showed a novel role for SPECC1L in guided movement through modulation of PI3K-AKT signaling.

scholarly journals SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jeremy P. Goering ◽  
Dona G. Isai ◽  
Everett G. Hall ◽  
Nathan R. Wilson ◽  
Edina Kosa ◽  
...  
Keyword(s):  
Wound Repair ◽  
Cleft Lip ◽  
Cell Movement ◽  
Live Imaging ◽  
Primary Role ◽  
Palatal Shelf ◽  
Collective Movement ◽  
Primary Mouse ◽  
Mutant Mouse Embryos ◽  
Mesenchyme Cells

AbstractCleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.

scholarly journals Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle, migration, and lineage selection

Methods ◽  
2018 ◽  
Vol 133 ◽  
pp. 81-90 ◽  
Author(s):  
Katja M. Piltti ◽  
Brian J. Cummings ◽  
Krystal Carta ◽  
Ayla Manughian-Peter ◽  
Colleen L. Worne ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Neural Stem Cells ◽  
Single Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Live Cell ◽  
Time Lapse Imaging

scholarly journals Role of fibronectin in primary mesenchyme cell migration in the sea urchin.

1985 ◽  
Vol 101 (4) ◽  
pp. 1487-1491 ◽  
Author(s):  
H Katow ◽  
M Hayashi
Keyword(s):  
Cell Migration ◽  
Sea Urchin ◽  
Culture Media ◽  
Horse Serum ◽  
Time Lapse ◽  
Mesenchyme Cell ◽  
Mesenchyme Cells ◽  
Primary Mesenchyme Cells

We studied the effect of fibronectin (FN) on the behavior of primary mesenchyme cells isolated from sea urchin mesenchyme blastulae in vitro using a time-lapse technique. The migration of isolated primary mesenchyme cells reconstituted in seawater and horse serum is dependent on the presence or absence of exogenous FN in the culture media. The cells in FN, 4 and 40 micrograms/ml, show a high percentage of migration and migrate long distances, whereas a higher concentration of FN at 400 micrograms/ml tends to inhibit migration.

Time-Lapse Video Microscopy and Single Cell Tracking to Study Neural Cell Behavior In Vitro

2019 ◽  
pp. 183-194
Author(s):  
Lucía Paniagua-Herranz ◽  
Rosa Gómez-Villafuertes ◽  
David de Agustín-Durán ◽  
Sergio Gascón ◽  
Raquel Pérez-Sen ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Tracking ◽  
Time Lapse ◽  
Neural Cell ◽  
Video Microscopy ◽  
Cell Behavior ◽  
Time Lapse Video

scholarly journals Capturing T Lymphocytes’ Dynamic Interactions With Human Neural Cells Using Time-Lapse Microscopy

2021 ◽  
Vol 12 ◽  
Author(s):  
Florent Lemaître ◽  
Ana Carmena Moratalla ◽  
Negar Farzam-kia ◽  
Yves Carpentier Solorio ◽  
Olivier Tastet ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
T Lymphocyte ◽  
Cell Tracking ◽  
Time Lapse ◽  
Neural Cells ◽  
Visual Interpretation ◽  
Time Lapse Microscopy ◽  
Multiple Variables

To fully perform their functions, T lymphocytes migrate within organs’ parenchyma and interact with local cells. Infiltration of T lymphocytes within the central nervous system (CNS) is associated with numerous neurodegenerative disorders. Nevertheless, how these immune cells communicate and respond to neural cells remains unresolved. To investigate the behavior of T lymphocytes that reach the CNS, we have established an in vitro co-culture model and analyzed the spatiotemporal interactions between human activated CD8+ T lymphocytes and primary human astrocytes and neurons using time-lapse microscopy. By combining multiple variables extracted from individual CD8+ T cell tracking, we show that CD8+ T lymphocytes adopt a more motile and exploratory behavior upon interacting with astrocytes than with neurons. Pretreatment of astrocytes or neurons with IL-1β to mimic in vivo inflammation significantly increases CD8+ T lymphocyte motility. Using visual interpretation and analysis of numerical variables extracted from CD8+ T cell tracking, we identified four distinct CD8+ T lymphocyte behaviors: scanning, dancing, poking and round. IL-1β-pretreatment significantly increases the proportion of scanning CD8+ T lymphocytes, which are characterized by active exploration, and reduces the proportion of round CD8+ T lymphocytes, which are less active. Blocking MHC class I on astrocytes significantly diminishes the proportion of poking CD8+ T lymphocytes, which exhibit synapse-like interactions. Lastly, our co-culture time-lapse model is easily adaptable and sufficiently sensitive and powerful to characterize and quantify spatiotemporal interactions between human T lymphocytes and primary human cells in different conditions while preserving viability of fragile cells such as neurons and astrocytes.

TGF-β isoform release and activation during in vitro bronchial epithelial wound repair

2002 ◽  
Vol 282 (1) ◽  
pp. L115-L123 ◽  
Author(s):  
William J. Howat ◽  
Stephen T. Holgate ◽  
Peter M. Lackie
Keyword(s):  
Wound Repair ◽  
Transforming Growth Factor ◽  
Time Lapse ◽  
Normal Function ◽  
Inhibition Of Proliferation ◽  
Bronchial Epithelial ◽  
Biological Damage ◽  
Environmental Agents ◽  
Tgf Β1

Restitution of an epithelial layer after environmental or biological damage is important to maintain the normal function of the respiratory tract. We have investigated the role of transforming growth factor (TGF)-β isoforms in the repair of layers of 16HBE 14o− bronchial epithelial-derived cells after damage by multiple scoring. ELISA showed that both latent TGF-β1 and TGF-β2 were converted to their active forms 2 h after wounding. Time-lapse microscopy showed that the addition of TGF-β1, but not TGF-β2, progressively increased the rate of migration of damaged monolayers at concentrations down to 250 pg/ml. This increase was blocked by addition of a neutralizing TGF-β1 antibody. Phase-contrast microscopy and inhibition of proliferation with mitomycin C showed that proliferation was not required for migration. These results demonstrate that conversion of latent to active TGF-β1 and TGF-β2 during in vitro epithelial wound repair occurs quickly and that TGF-β1 speeds epithelial repair. A faster repair may be advantageous in preventing access of environmental agents to the internal milieu of the lung although the production of active TGF-β molecules may augment subepithelial fibrosis.

scholarly journals Insulin and IGF-I Inhibit GH Synthesis and Release in Vitro and in Vivo by Separate Mechanisms

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2410-2420 ◽  
Author(s):  
Manuel D. Gahete ◽  
José Córdoba-Chacón ◽  
Qing Lin ◽  
Jens C. Brüning ◽  
C. Ronald Kahn ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Inhibitory Effect ◽  
Pituitary Cell ◽  
Primary Role ◽  
Gh Secretion ◽  
Relative Contribution ◽  
Primary Mouse ◽  
Igf I

Abstract IGF-I is considered a primary inhibitor of GH secretion. Insulin may also play an important role in regulating GH levels because insulin, like IGF-I, can suppress GH synthesis and release in primary pituitary cell cultures and insulin is negatively correlated with GH levels in vivo. However, understanding the relative contribution insulin and IGF-I exert on controlling GH secretion has been hampered by the fact that circulating insulin and IGF-I are regulated in parallel and insulin (INSR) and IGF-I (IGFIR) receptors are structurally/functionally related and ubiquitously expressed. To evaluate the separate roles of insulin and IGF-I in directly regulating GH secretion, we used the Cre/loxP system to knock down the INSR and IGFIR in primary mouse pituitary cell cultures and found insulin-mediated suppression of GH is independent of the IGFIR. In addition, pharmacological blockade of intracellular signals in both mouse and baboon cultures revealed insulin requires different pathways from IGF-I to exert a maximal inhibitory effect on GH expression/release. In vivo, somatotrope-specific knockout of INSR (SIRKO) or IGFIR (SIGFRKO) increased GH levels. However, comparison of the pattern of GH release, GH expression, somatotrope morphometry, and pituitary explant sensitivity to acute GHRH challenge in lean SIRKO and SIGFRKO mice strongly suggests the primary role of insulin in vivo is to suppress GH release, whereas IGF-I serves to regulate GH synthesis. Finally, SIRKO and/or SIGFRKO could not prevent high-fat, diet-induced suppression of pituitary GH expression, indicating other factors/tissues are involved in the decline of GH observed with weight gain.

scholarly journals Linoleic and oleic acids enhance cell migration by altering the dynamics of microtubules and the remodeling of the actin cytoskeleton at the leading edge

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Masner ◽  
N. Lujea ◽  
M. Bisbal ◽  
C. Acosta ◽  
Patricia Kunda
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Cancer Cell ◽  
Cell Tracking ◽  
Leading Edge ◽  
Time Lapse ◽  
Control Group ◽  
Microtubule Organizing Center ◽  
Actin Waves

AbstractFatty acids (FA) have a multitude of biological actions on living cells. A target of their action is cell motility, a process of critical importance during cancer cell dissemination. Here, we studied the effect of unsaturated FA on ovarian cancer cell migration in vitro and its role in regulating cytoskeleton structures that are essential for cell motility. Scratch wound assays on human ovary cancer SKOV-3 cell monolayers revealed that low doses (16 μM) of linoleic acid (LA, 18:2 ω6) and oleic acid (OA; 18:1 ω9) promoted migration, while α-linolenic acid (ALA, 18:3 ω3), showed a migration rate similar to that of the control group. Single cell tracking demonstrated that LA and OA-treated cells migrated faster and were more orientated towards the wound closure than control. In vitro addition of those FA resulted in an increased number, length and protrusion speed of filopodia and also in a prominent and dynamic lamellipodia at the cell leading edge. Using time-lapse video-microscopy and FRAP we observed an increase in both the speed and frequency of actin waves associated with more mobile actin and augmented Rac1 activity. We also observed that FA induced microtubule-organizing center (MTOC)-orientation towards the cell front and affected the dynamics of microtubules (MT) in the direction of cell migration. We propose that environmental cues such as OA and LA present in ascitic fluid, should be taken into account as key factors for the regulation of cell migration.

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