Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena

Zinchuk, Vadim S.; Zinchuk, Olga; Okada, Teruhiko

doi:10.1267/ahc.07002

Cited by 377 publications

(325 citation statements)

References 33 publications

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“…Briefly, separate volumes were defined for LROs (based on Lamp staining) and GrB by automatic thresholding based on absolute intensity. Overlap coefficients according to Manders, 51 which range from 0 (no colocalisation) to 1 (100% colocalisation) and represent the true degree of colocalisation, were determined.…”

Section: Methodsmentioning

confidence: 99%

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

et al. 2014

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Cytotoxic lymphocytes (CLs) contain lysosome-related organelles (LROs) that perform the normal degradative functions of the lysosome, in addition to storage and release of powerful cytotoxins employed to kill virally infected or abnormal cells. Among these cytotoxins is granzyme B (GrB), a protease that has also been implicated in activation (restimulation)-induced cell death of natural killer (NK) and T cells, but the underlying mechanism and its regulation are unclear. Here we show that restimulation of previously activated human or mouse lymphocytes induces lysosomal membrane permeabilisation (LMP), followed by GrB release from LROs into the CL cytosol. The model lysosomal stressors sphingosine and Leu-Leu-methyl-ester, and CLs from gene-targeted mice were used to show that LMP releases GrB in both a time-and concentration-dependent manner, and that the liberated GrB is responsible for cell death. The endogenous GrB inhibitor Serpinb9 (Sb9) protects CLs against LMP-induced death but is decreasingly effective as the extent of LMP increases. We also used these model stressors to show that GrB is the major effector of LMP-mediated death in T cells, but that in NK cells additional effectors are released, making GrB redundant. We found that limited LMP and GrB release occurs constitutively in proliferating lymphocytes and in NK cells engaged with targets in vitro. In Ectromelia virus-infected lymph nodes, working NK cells lacking Sb9 are more susceptible to GrB-mediated death. Taken together, these data show that a basal level of LMP occurs in proliferating and activated lymphocytes, and is increased on restimulation. LMP releases GrB from LROs into the lymphocyte cytoplasm and its ensuing interaction with Sb9 dictates whether or not the cell survives. The GrB-Sb9 nexus may therefore represent an additional mechanism of limiting lymphocyte lifespan and populations.

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Section: Methodsmentioning

confidence: 99%

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

et al. 2014

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“…2E). Using Manders' colocalization coefficiency (21), above and below the endothelium, revealed a significant decrease in the degree of overlap specifically within the transmigrated part of the cell (Fig. 2F).…”

Section: Polarized Shedding Of L-selectin Occurs In Primary Human Monmentioning

confidence: 98%

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

Rzeniewicz

Newe

Gallardo

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

103

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L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/ CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.

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“…Images were acquired with a confocal microscope (Olympus, Japan). To determine the colocalization level of A 1 R and EGFR, Pearson's and Manders' coefficients were calculated using FV10-ASW2.1 software (Olympus) according to the previously reported analysis methods [23] . Briefly, Pearson's correlation coefficient describes the correlation of the intensity distribution between the channels, ranging from -1.0 to 1.0, where 0 indicates no significant correlation.…”

Section: Western Blot Analysismentioning

confidence: 99%

Paeoniflorin ameliorates ischemic neuronal damage in vitro via adenosine A1 receptor-mediated transactivation of epidermal growth factor receptor

Zhong

Song

et al. 2015

Acta Pharmacol Sin

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Aim: Paeoniflorin from Chinese herb Paeoniae Radix has been shown to ameliorate middle cerebral artery occlusion-induced ischemia in rats. The aim of this study was to investigate the mechanisms underlying the neuroprotective action of PF in cultured rat cortical neurons. Methods: Primary cultured cortical neurons of rats were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) insult. Cell survival was determined using MTT assay. HEK293 cells stably transfected with A 1 R (HEK293/A 1 R) were used for detailed analysis. Phosphorylation of the signaling proteins was evaluated by Western blot or immunoprecipitation. Receptor interactions were identified using co-immunoprecipitation and immunofluorescence staining. Results: Paeoniflorin (10 nmol/L to 1 μmol/L) increased the survival of neurons subjected to OGD/R. Furthermore, paeoniflorin increased the phosphorylation of Akt and ERK1/2 in these neurons. These effects were blocked by PI3K inhibitor wortmannin or MEK inhibitor U0126. Paeoniflorin also increased the phosphorylation of Akt and ERK1/2 in HEK293/A 1 R cells. Both A 1 R antagonist DPCPX and EGFR inhibitor AG1478 not only blocked paeoniflorin-induced phosphorylation of ERK1/2 and Akt in HEK293/A 1 R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. In addition, paeoniflorin increased the phosphorylation of Src kinase and activation of MMP-2 in HEK293/A 1 R cells. Both Src inhibitor PP2 and MMP-2/MMP-9 inhibitor BiPs not only blocked paeoniflorininduced phosphorylation of ERK1/2 (and Akt) in HEK293/A 1 R cells, but also paeoniflorin-increased survival of neurons subjected to OGD/R. Conclusion: Paeoniflorin promotes the survival of cultured cortical neurons by increasing Akt and ERK1/2 phosphorylation via A 1 Rmediated transactivation of EGFR.

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Quantitative Colocalization Analysis of Multicolor Confocal Immunofluorescence Microscopy Images: Pushing Pixels to Explore Biological Phenomena

Cited by 377 publications

References 33 publications

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

The granzyme B-Serpinb9 axis controls the fate of lymphocytes after lysosomal stress

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

Paeoniflorin ameliorates ischemic neuronal damage in vitro via adenosine A1 receptor-mediated transactivation of epidermal growth factor receptor

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