Cardiomyocyte binucleation is associated with aberrant mitotic microtubule distribution, mislocalization of RhoA and IQGAP3, as well as defective actomyosin ring anchorage and cleavage furrow ingression

2018 ◽  
Vol 114 (8) ◽  
pp. 1115-1131 ◽  
Author(s):  
Marina Leone ◽  
Gentian Musa ◽  
Felix Benedikt Engel
Keyword(s):  
Time Lapse ◽  
Equatorial Region ◽  
Central Component ◽  
Cleavage Furrow ◽  
Cardiomyocyte Proliferation ◽  
The Difference ◽  
Time Lapse Imaging ◽  
Regenerative Therapies

Abstract Aims After birth mammalian cardiomyocytes initiate a last cell cycle which results in binucleation due to cytokinesis failure. Despite its importance for cardiac regenerative therapies, this process is poorly understood. Here, we aimed at a better understanding of the difference between cardiomyocyte proliferation and binucleation and providing a new tool to distinguish these two processes. Methods and results Monitoring of cell division by time-lapse imaging revealed that rat cardiomyocyte binucleation stems from a failure to properly ingress the cleavage furrow. Astral microtubule required for actomyosin ring anchorage and thus furrow ingression were not symmetrically distributed at the periphery of the equatorial region during anaphase in binucleating cardiomyocytes. Consequently, RhoA, the master regulator of actomyosin ring formation and constriction, non-muscle myosin IIB, a central component of the actomyosin ring, as well as IQGAP3 were abnormally localized during cytokinesis. In agreement with improper furrow ingression, binucleation in vitro and in vivo was associated with a failure of RhoA and IQGAP3 to localize to the stembody of the midbody. Conclusion Taken together, these results indicate that naturally occurring cytokinesis failure in primary cardiomyocytes is due to an aberrant mitotic microtubule apparatus resulting in inefficient anchorage of the actomyosin ring to the plasma cell membrane. Thus, cardiomyocyte binucleation and division can be discriminated by the analysis of RhoA as well as IQGAP3 localization.

Enamel Demineralization and Remineralization Detection Using Non-invasive Optical Imaging

2021 ◽  
Vol 15 (9) ◽  
pp. 2766-2769
Author(s):  
Hijab Fatemah Memon ◽  
Suraiya Hirani ◽  
Jaweria Yousfani ◽  
Reema Aslam ◽  
Sehar Mushtaque ◽  
...  
Keyword(s):  
Time Lapse ◽  
Signal Acquisition ◽  
Scattering Media ◽  
Lesion Depth ◽  
Promising Tool ◽  
The Difference ◽  
Imagej Software ◽  
B Scan Images

Introduction: Optical Coherence Tomography (OCT), is one of the most emerging diagnostic imaging technique. It is capable of producing 3D images using optical scattering media. The fast signal acquisition quality has made it a promising tool to detect early in vivo and in vitro lesions. The aim of this study was to reproduce previous demineralization results and to detect remineralization using OCT. Methodology: Bovine enamel discs were used thoroughly in this study. The study was done using the flow cell for detecting demineralization and remineralization following 96 hours demineralization and 192 hours remineralization. A time lapse monitoring was done and the lesions were assessed visually. ImageJ software was used to process the images produced through OCT. The lesion depth and intensity was measured across the images produced which helped in assessing the difference between remineralization and demineralization. Results: OCT B-scan images result in increased backscattering light which is considered the main principle to measure lesion depth and mineral loss. Whereas, in remineralization decreased band of light appeared with reduction in porosity during mineral precipitation. The results for remineralization were diverse and could not be assessed. Conclusion: OCT is favorable technique to detect demineralization and remineralization but it still needs a lot of improvement especially regarding remineralization there are limitations which need to be improved.

scholarly journals Sindbis Virus-Induced Neuronal Death Is both Necrotic and Apoptotic and Is Ameliorated byN-Methyl-d-Aspartate Receptor Antagonists

2001 ◽  
Vol 75 (15) ◽  
pp. 7114-7121 ◽  
Author(s):  
Jennifer L. Nargi-Aizenman ◽  
Diane E. Griffin
Keyword(s):  
Cell Death ◽  
Cortical Neurons ◽  
Sindbis Virus ◽  
Apoptotic Cell ◽  
Time Lapse ◽  
Apoptotic Cell Death ◽  
Time Lapse Imaging ◽  
Alphavirus Infection

ABSTRACT Virus infection of neurons leads to different outcomes ranging from latent and noncytolytic infection to cell death. Viruses kill neurons directly by inducing either apoptosis or necrosis or indirectly as a result of the host immune response. Sindbis virus (SV) is an alphavirus that induces apoptotic cell death both in vitro and in vivo. However, apoptotic changes are not always evident in neurons induced to die by alphavirus infection. Time lapse imaging revealed that SV-infected primary cortical neurons exhibited both apoptotic and necrotic morphological features and that uninfected neurons in the cultures also died. Antagonists of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors protected neurons from SV-induced death without affecting virus replication or SV-induced apoptotic cell death. These results provide evidence that SV infection activates neurotoxic pathways that result in aberrant NMDA receptor stimulation and damage to infected and uninfected neurons.

Forming Cytoplasmic Fragment Is Implicated In the Final Step of Platelet Production

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4793-4793
Author(s):  
Shugo Kowata ◽  
Kazunori Murai ◽  
Kenichi Nomura ◽  
Tatsuo Oyake ◽  
Shigeki Ito ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Time Lapse ◽  
In Vitro Assays ◽  
Spatial Continuity ◽  
Platelet Production ◽  
Microscopic Evaluation ◽  
Large Particles ◽  
Time Lapse Imaging

Abstract Abstract 4793 Introduction: Conventional microscopic evaluation of bone marrow (BM) and in vitro assays have suggested that platelets arise from the proplatelet (PP) that extend from the mature megakaryocytes (MKs) in BM. On the other hand, recently, a study with in vivo imaging showed that MKs routinely release heterogeneous substantial large particles into BM sinusoids (Tobias Junt et. al., Science 317,1767, 2007). They noted that large particles may represent multiple intertwined or single immature proplatelets. However, it has been unclear whether the heterogeneous large particles consist of intertwined strings PPs or “cytoplasmic fragment (CF)”. Thus, in our study, to resolve this riddle, we planed to observe the dynamics of MKs with a modified imaging technique and we have cleared the presence and role of CF in platelet production. Materials and methods: Our study was approved by the Iwate Medical University Institutional Animal Care and Use Committee. 1) Mice: Six- to 8-week-old transgenic C57/BL6 (actin promoter driven EGFP) was used. 2) In vitro time lapse imaging of MK and PPF study: Primary mature MKs from femur BM were cultured for 12 hrs. Time lapse images were taken using Zeiss LSM510 meta confocal microscope (CLM). 3) 3-D reconstitution imaging of fixed BM study: the BM core was removed and immediately fixed and stained with PE-conjugated anti-CD61 antibody. Images were taken using CLM and reconstituted to 3-D images to keep the continuity between MK cell body and PP. 4) BM imaging by Multiphoton intravital microscopy (MP-IVM): Mice were anesthetized, and the frontoparietal skull was exposed. To trace individual MK over time in BM of living mice, time lapse images were taken. Results: By in vitro time lapse imaging of MK study, it become clear that primary cultured MK formed CF in which morphology was distinctly different from PP (Fig.1). Reversible interconversion between CF and PP was observed also. We observed that CF formation was more augmented in the presence of other BM cells. Because 3-D reconstitution imaging of fixed BM study has a benefit to observe amorphous structure without breaking of spatial continuity, we successfully proved the presence of CF and PP in BM sinusoid clearly (Fig.2). BM imaging by MP-IVM demonstrated that MK formed CF and extended protrusions into sinusoids. We have proved that MK formed and extended CF and PP coincidentally into sinusoids (Fig.3). Discussion: We had taken an evidence of presence of CF by in vitro time lapse imaging and 3-D reconstitution imaging. The meaning of reversible interconversion between CF and PP remains unclear in our study. This fact may closely associate with the efficacy of platelet production and avoiding precocious platelet activation in BM. The result that MK produced and extended CF and PP coincidentally suggests that both PP and CF formation may be essential for platelet production process. In conclusion, MK forms PP and CF in living BM. Both PP and CF have critical roles in platelet production mechanism. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Quantifying endothelial cell proliferation in the zebrafish embryo

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 1032
Author(s):  
George Bowley ◽  
Timothy JA Chico ◽  
Jovana Serbanovic-Canic ◽  
Paul C Evans
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Zebrafish Embryo ◽  
Time Lapse ◽  
Endothelial Cell Proliferation ◽  
Small Scale ◽  
Zebrafish Embryos ◽  
Time Lapse Imaging

Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.

scholarly journals Actin Filament Assembly by Myristoylated, Alanine-rich C Kinase Substrate–Phosphatidylinositol-4,5-diphosphate Signaling Is Critical for Dendrite Branching

2008 ◽  
Vol 19 (11) ◽  
pp. 4804-4813 ◽  
Author(s):  
Haimin Li ◽  
Gang Chen ◽  
Bing Zhou ◽  
Shumin Duan
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Time Lapse ◽  
Dependent Manner ◽  
Filamentous Actin ◽  
Kinase Substrate ◽  
Extensive Growth ◽  
Time Lapse Imaging

Dendrites undergo extensive growth and branching at early stages, but relatively little is known about the molecular mechanisms underlying these processes. Here, we show that increasing the level of myristoylated, alanine-rich C kinase substrate (MARCKS), a prominent substrate of protein kinase C and a phosphatidylinositol-4,5-diphosphate [PI(4,5)P2] sequestration protein highly expressed in the brain, enhanced branching and growth of dendrites both in vitro and in vivo. Conversely, knockdown of endogenous MARCKS by RNA interference reduced dendritic arborization. Results from expression of different mutants indicated that membrane binding is essential for MARCKS-induced dendritic morphogenesis. Furthermore, MARCKS increased the number and length of filamentous actin-based filopodia along neurites, as well as the motility of filopodia, in a PI(4,5)P2-dependent manner. Time-lapse imaging showed that MARCKS increased frequency of filopodia initiation but did not affect filopodia longevity, suggesting that MARCKS may increase dendritic branching through its action on filopodia initiation. These findings demonstrate a critical role for MARCKS–PI(4,5)P2 signaling in regulating dendrite development.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

Reliability of a Single β-Thromboglobulin Measurement in a Diabetic Population : Importance of PGE1 in Anticoagulant Mixture

1987 ◽  
Vol 57 (02) ◽  
pp. 201-204 ◽  
Author(s):  
P Y Scarabin ◽  
L Strain ◽  
C A Ludlam ◽  
J Jones ◽  
E M Kohner
Keyword(s):  
In Vitro Release ◽  
Mean Value ◽  
Reliable Estimate ◽  
Diabetic Patients ◽  
Single Measurement ◽  
Diabetic Population ◽  
The Difference ◽  
Vitro Release

SummaryDuring the collection of samples for plasma β-thromboglobulin (β-TG) determination, it is well established that artificially high values can be observed due to in-vitro release. To estimate the reliability of a single β-TG measurement, blood samples were collected simultaneously from both arms on two separate occasions in 56 diabetic patients selected for a clinical trial. From each arm, blood was taken into two tubes containing an anticoagulant mixture with (tube A) and without (tube B) PGE!. The overall mean value of B-TG in tube B was 1.14 times higher than in tube A (p <0.01). The markedly large between-arms variation accounted for the most part of within-subject variation in both tubes and was significantly greater in tube B than in tube A. Based on the difference between B-TG values from both arms, the number of subjects with artifically high B-TG values was significantly higher in tube B than in tube A on each occasion (overall rate: 28% and 14% respectively). Estimate of between-occasions variation showed that B-TG levels were relatively stable for each subject between two occasions in each tube. It is concluded that the use of PGEi decreases falsely high B-TG levels, but a single measurement of B-TG does not provide a reliable estimate of the true B-TG value in vivo.

scholarly journals Cytokinesis and Midzone Microtubule Organization inCaenorhabditis elegans Require the Kinesin-like Protein ZEN-4

1998 ◽  
Vol 9 (8) ◽  
pp. 2037-2049 ◽  
Author(s):  
William B. Raich ◽  
Adrienne N. Moran ◽  
Joel H. Rothman ◽  
Jeff Hardin
Keyword(s):  
Cell Division ◽  
Null Allele ◽  
Time Lapse ◽  
Equatorial Region ◽  
Microtubule Organization ◽  
Dividing Cells ◽  
Spindle Midzone ◽  
Cross Links ◽  
Complete Cell

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele ofzen-4, an MKLP1 homologue in the nematodeCaenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

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