Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Abstract

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes. Video LinkThe video component of this article can be found at

Non-Overlapping Distributions and Functions of the VDAC Family in Ciliogenesis

“…Using a VDAC1-specific siRNA (siVDAC1), we are able to consistently deplete more than 80% of total VDAC1 protein in siVDAC1 cells, compared to control cells (siControl, Figure 3 C). siVDAC1 also reduced the γ-tubulin-normalized centrosomal level of VDAC1 more than two-fold ( Figure 3 C), as determined by our modified quantitative immunofluorescence assay [ 30 ]. To ensure we compared centrosomal VDAC1 levels between cells at similar stages of the cell cycle, we restricted the analysis to cells that were in S-phase as judged by incorporation of BrdU; S-phase also represents the strongest VDAC1 signal at centrosomes (see below).…”

“…Click-iT EdU Cell Proliferation kit (Invitrogen) was used according to manufacturer’s instruction to visualize EdU-positive cells. The centrosomal level of VDAC1 was measured as a ratio of the total fluorescence signal of VDAC1 to that of γ-tubulin at centrosomes using our recently described quantitative IIF technique [ 30 ]. BrdU-positive cells from both samples were imaged under identical conditions for the analysis.…”

Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function

“…HeLa cells transfected with GFP or GFP-Cetn3 were arrested in S phase with a 24-h HU treatment, and cell pairings in which one cell expressed GFP or GFP-Cetn3 and the other did not were analyzed (e.g., Figure 10A ). We then determined the ratio of centrosomal pT676 (normalized to γ-tubulin; F pT676 ) in the GFP-positive cell to that in the untransfected cell for 25 pairs from each transfection, as previously described ( Kasbek et al ., 2007 ; Majumder and Fisk, 2014 ). Whereas centrosomal pT676 staining in cells expressing GFP alone was essentially the same as in untransfected cells (the median value of F pT676 (GFP+)/ F pT676 (GFP–) was close to 1), there was a twofold reduction in centrosomal pT676 in cells expressing GFP-Cetn3 ( Figure 10B ), and the difference between GFP and GFP-Cetn3 was highly significant ( p < 0.0001).…”

“…, 2010 ), rabbit anti-CP110 (this study), mouse anti–γ-tubulin (GTU-88; Sigma-Aldrich), rabbit anti–γ-tubulin (Sigma-Aldrich), goat anti–γ-tubulin (Santa Cruz Biotechnology), rabbit anti-Cep135 (Abcam), mouse anti–acetylated tubulin (Ac-tub; Sigma-Aldrich), DM1A mouse anti–α-tubulin (Sigma-Aldrich), pan Mps1 (H00007272-M02; Novus Biologicals, Littleton, CO), rat anti-BrdU (Abcam), and mouse anti-GFP (Life Technologies, Carlsbad, CA). The intensity of total centrosomal Mps1 protein (pan Mps1 antibody MO2) or centrosomal Mps1 activity (phosphospecific Mps1 antibody pT676) was measured in 25 cells for each sample as previously described ( Majumder et al ., 2012 ; Majumder and Fisk, 2014 ). A rabbit antibody against Cetn3 was generated by injecting 6-histidine (His)-Cetn3 into rabbits (Lampire Biologicals, Pipersville, PA), and then affinity purification of serum against His-Cetn3 bound to Affigel 15 (Bio-Rad, Hercules, CA) was performed as previously described for Cetn2 ( Yang et al.…”

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

“…In recent years many studies have developed different quantitative microscopy assays for fixed cells. 42 Progress in centromere-kinetochore biology often requires an understanding of the centromere-specific or kinetochore-specific function of proteins of which subcellular spatialtemporal regulation reflects the changing functions of these proteins during cell cycle. Therefore, here we developed methods of IIF staining and a quantitative IIF assay to specifically analyze the relative levels of endogenous and exogenous CENP-A proteins, which can be applicable in differently treated samples.…”