Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images

Zinchuk, Vadim S.; Grossenbacher-Zinchuk, Olga

doi:10.1002/0471143030.cb0419s52

Cited by 59 publications

(8 citation statements)

References 28 publications

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“…The lamellipodia was segmented from the rest of the cell and scatterplots of both signals were made to show signal distribution. We used Mander's overlap coefficient, which takes into account the contribution of each fluorophore to the colocalized signals [70], to measure colocalization of F-actin and Arp2/3 in the cell interior and at the lamellipodia.…”

Section: Quantification Of Retrograde Flowmentioning

confidence: 99%

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

et al. 2020

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Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.

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Section: Quantification Of Retrograde Flowmentioning

confidence: 99%

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

et al. 2020

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“…Co-localization was evaluated on medial optical sections using NIS-Elements software (Nikon, Nikon, Melville, NY, USA). Overlap coefficient k2, sensitive to differences in intensity for red signals [22,23], was calculated according to Manders et al [24].…”

Section: Immunofluorescence and Confocal Analysismentioning

confidence: 99%

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations

Antoniel

Traina

Merlini

et al. 2020

Cells

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Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

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“…Image analysis was performed using ImageJ Soware and Simple PCI soware. Colocalization coefficients 81,82 were calculated using ImageJ plugin Colocalization Indices. Confocal uorescence images were taken on a Zeiss LSM 710 confocal microscope using an X63 (NA 1.40) objective and 405 nm laser excitation.…”

Section: Fluorescence Imaging and Study Of The Effect Of Temperature mentioning

confidence: 99%

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques

Clède

Lambert

Sandt

et al. 2013

Analyst

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3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

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Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images

Cited by 59 publications

References 28 publications

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques

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