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

Summary

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.

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.

Introduction

Centrosomes consist of a pair of centrioles surrounded by pericentriolar material (PCM). Being the major microtubule organizing centers (MTOCs) in mammalian cells, centrosomes serve as the two poles of mitotic spindles in dividing cells, and thus help maintain genomic integrity¹. In quiescent cells (e.g., during G0 phase), one of the two centrioles of the centrosome, namely the mother centriole, is transformed into a basal body to assemble the primary cilium, a sensory organelle protruding out from the cell surface². Once the cells re-enter the cell cycle, primary cilia are disassembled and each centriole directs the assembly of a procen....

Protocol

1. Cell Culture

1. Use human telomerase reverse transcriptase (hTERT) immortalized retinal pigment epithelial cells (hTERT-RPE1; referred to here as RPE1).
   NOTE: RPE1 cells are near-diploid, non-transformed human cells that are commonly used to study centriole assembly and ciliogenesis. These cells follow a normal centriole duplication cycle coordinated with a regulated cell cycle.
2. Passage a near-confluent 100 mm cell culture dish of RPE1 cells at 1:5 dilution of the original culture into a fresh 100 mm dish containing 10 ml of DME/F-12 (1:1) medium supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml penicillin G and 100 µg/ml strep....

Results

Our recent studies identified novel centrosomal localization and function of VDAC3, one of the mitochondrial porins^16,28. Immunostaining of several mammalian cells including RPE1 cells using a VDAC3-specific antibody showed prominent centrosomal staining and comparatively weak mitochondrial staining. We also showed that centrosomal VDAC3 is preferentially associated with the mother centriole, and the centrosomal pool of both the endogenous and ectopically expressed VDAC3 is regulated by degradation¹⁶

Discussion

Quantitative microscopy in cell biology is commonly associated with live-cell imaging assays such as Fluorescence Resonance Energy Transfer (FRET), Fluorescence Recovery After Photobleaching (FRAP), etc. However, there are growing examples of cell biologists developing different quantitative microscopy assays for fixed cells in recent years^27,34-36. Importantly, progress in understanding centrosome biology often requires understanding of the centrosome-specific function of proteins whose centrosomal p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fibronectin	Sigma	F8141	Stock of 1 mg/ml in water
DMSO	Sigma	D2650
MG115	Sigma	SCP0005	Stock of 10 mM in DMSO
BrdU	Sigma	B5002	Stock of 10 mM in DMSO
Anti-γ-tubulin (mouse monoclonal, clone GTU 88)	Sigma	T 6557	1:200 in IIF blocking buffer
Anti-VDAC3 (rabbit polyclonal)	Aviva Systems Biology	ARP35180-P050	1:50 in IIF blocking buffer, 1:1,000 in WB blocking buffer
Anti-Sas6 (mouse monoclonal)	Santa cruz biotechnology	sc-81431	1:100 in IIF blocking buffer
Anti-Cep135 (rabbit polyclonal)	Abcam	ab-75005	1:500 in IIF blocking buffer
Anti-BrdU (rat monoclonal)	Abcam	ab6326	1:250 in IIF blocking buffer
Alexa Fluor 350 Goat Anti-Rat IgG (H+L)	Life technologies	A21093	1:200 in IIF blocking buffer
Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody	Life technologies	A21202	1:1,000 in IIF blocking buffer
Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody	Life technologies	A21203	1:1,000 in IIF blocking buffer
Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody	Life technologies	A21206	1:1,000 in IIF blocking buffer
Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody	Life technologies	A21207	1:1,000 in IIF blocking buffer
Anti-γ-tubulin (rabbit polyclonal)	Sigma	T5192	1:1,000 in WB blocking buffer
Anti-α-tubulin (mouse monoclonal, DM1A)	Sigma	T9026	1:20,000 in WB blocking buffer
Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L)	Life technologies	A10043	1:10,000 in WB blocking buffer
Mouse IgG (H&L) Antibody IRDye800CW Conjugated	Rockland antibodies	610-731-002	1:10,000 in WB blocking buffer
SlowFade Gold Antifade Reagent	Life technologies	S36936	Mounting media
Round coverslips 12CIR.-1	Fisherbrand	12-545-80
Olympus IX-81 microscope	Olympus
Retiga ExiFAST 1394 IR camera	QImaging	32-0082B-238
100X Plan Apo oil immersion objective	Olympus		1.4 numerical aperture
Slidebook software package	Intelligent Imaging Innovations
Odyssey IR Imaging System	Li-cor Biosciences
Bicinchoninic acid (BCA) assay	Thermo Scientific	23227
U-MNU2 Narrow UV cube	Olympus	U-M622	Filter
U-MNU2 Narrow Blue cube	Olympus	U-M643	Filter
U-MNU2 Narrow Green cube	Olympus	U-M663	Filter