Abstract:Immunocytochemistry is a powerful tool for detection and visualization of specific molecules in living or fixed cells, their localization and their relative abundance. One of the most commonly used fluorescent DNA dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used extensively for visualizing cell nuclei. It is excited by UV light and emits characteristic blue fluorescence. Here, we report a phenomenon based on an appa… Show more
“…We ruled out the possibility that the accumulation was the result of UV-induced photoconversion of Hoechst 33342 by confirming that there was no detectable increase in green fluorescence with normal photodamage conditions (355 nm UV laser at 1 nW for 500 ms) in the absence of GFP-fusion proteins. In fact, Hoechst 33342 was chosen because it exhibited the lowest level of UV-induced photoconversion compared with DAPI and Hoechst 33258 42 . Fig.…”
Phosphoinositide lipids (PPIs) are enriched in the nucleus and are accumulated at DNA damage sites. Here, we investigate roles of nuclear PPIs in DNA damage response by sequestering specific PPIs with the expression of nuclear-targeted PH domains, which inhibits recruitment of Ataxia telangiectasia and Rad3-related protein (ATR) and reduces activation of Chk1. PPI-binding domains rapidly (< 1 s) accumulate at damage sites with local enrichment of PPIs. Accumulation of PIP3 in complex with the nuclear receptor protein, SF1, at damage sites requires phosphorylation by inositol polyphosphate multikinase (IPMK) and promotes nuclear actin assembly that is required for ATR recruitment. Suppressed ATR recruitment/activation is confirmed with latrunculin A and wortmannin treatment as well as IPMK or SF1 depletion. Other DNA repair pathways involving ATM and DNA-PKcs are unaffected by PPI sequestration. Together, these findings reveal that nuclear PPI metabolism mediates an early damage response through the IPMK-dependent pathway to specifically recruit ATR.
“…We ruled out the possibility that the accumulation was the result of UV-induced photoconversion of Hoechst 33342 by confirming that there was no detectable increase in green fluorescence with normal photodamage conditions (355 nm UV laser at 1 nW for 500 ms) in the absence of GFP-fusion proteins. In fact, Hoechst 33342 was chosen because it exhibited the lowest level of UV-induced photoconversion compared with DAPI and Hoechst 33258 42 . Fig.…”
Phosphoinositide lipids (PPIs) are enriched in the nucleus and are accumulated at DNA damage sites. Here, we investigate roles of nuclear PPIs in DNA damage response by sequestering specific PPIs with the expression of nuclear-targeted PH domains, which inhibits recruitment of Ataxia telangiectasia and Rad3-related protein (ATR) and reduces activation of Chk1. PPI-binding domains rapidly (< 1 s) accumulate at damage sites with local enrichment of PPIs. Accumulation of PIP3 in complex with the nuclear receptor protein, SF1, at damage sites requires phosphorylation by inositol polyphosphate multikinase (IPMK) and promotes nuclear actin assembly that is required for ATR recruitment. Suppressed ATR recruitment/activation is confirmed with latrunculin A and wortmannin treatment as well as IPMK or SF1 depletion. Other DNA repair pathways involving ATM and DNA-PKcs are unaffected by PPI sequestration. Together, these findings reveal that nuclear PPI metabolism mediates an early damage response through the IPMK-dependent pathway to specifically recruit ATR.
“…An experimenter expecting that the DNA dyes emit in the blue range can misinterpret the green signal as that arising from another probe in the sample. This risk has been raised previously 1, 3, 6 , yet the artefact is rarely controlled for.…”
A commonly used approach for assessing DNA repair factor recruitment in mammalian cells is to induce DNA damage with a laser in the UV or near UV range and follow the local increase of GFP-tagged proteins at the site of damage. Often these measurements are performed in the presence of the blue DNA dye Hoechst, which is used as a photosensitizer. However, a light-induced switch of Hoechst from a blue-light to a green-light emitter will give a false positive signal at the site of damage. Thus, photoconversion signals must be subtracted from the overall green-light emission to determine true recruitment. Here we demonstrate the photoconversion effect and suggest control experiments to exclude false-positive results.
“…The rate of photoconversion of DAPI and similar dyes may vary depending on instrument configuration. While photoconversion of DAPI can become apparent after a few seconds [2], detection of intense photoconversion product fluorescence more commonly requires continuous UV irradiation for 30 seconds or longer [1,3,6]. Photoconversion artifacts can be avoided by keeping UV excitation of DAPI as brief as possible when locating and focusing on specimens.…”
Exposure of the nuclear counterstain DAPI to just a few seconds of UV excitation light during fluorescence imaging can photoconvert the dye to stable green-and red-emitting forms, resulting in imaging artifacts in multi-color staining experiments. This article reviews the published literature on photoconversion and compares the effect of different mounting media on photoconversion of DAPI in stained tissue sections. Once photoconversion is recognized as a potential source of non-specific fluorescence, careful imaging practices or reagent selection can be used to avoid it.
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