Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

Abstract

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

PML‐like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication‐based single‐strand breaks

“…4A). This observation agrees with the description of 3D organization of SP100-containing nuclear bodies, which was revealed by 4Pi superresolution microscopy (35) and other microscopy techniques (36). We also demonstrate that the investigated SP100-containing nuclear bodies are always localized in close vicinity to but outside regions of active DNA replication (Fig.…”

Membraneless nuclear organelles and the search for phases within phases

“…Early electron microscopy studies found that PML forms a peripheral ring around an RNA-deficient core (Boisvert et al, 2000). This was confirmed by SRM, which also indicated that the PML nuclear body core is filled with SUMOylated proteins, which act as a molecular glue to hold the structure together (Hands, Cuchet-Lourenco, Everett, & Hay, 2014;Hoischen, Monajembashi, Weisshart, & Hemmerich, 2018). The position of different PML protein isoforms varies within the structure, hinting at the existence of additional sub-phases (Hands et al, 2014).…”

The HSV-1 ubiquitin ligase ICP0: Modifying the cellular proteome to promote infection

“…ICP0 was initially identified as an E3 Ub ligase by virtue of its ability to localize to and disrupt PML-NBs ( Everett, 2000 ; Maul et al, 1993 ; Maul and Everett, 1994 ; Everett and Maul, 1994 ; Everett et al, 1998 ; Chelbi-Alix and de The, 1999 ). PML-NBs are highly dynamic nuclear substructures composed of more than 70 proteins that respond to a variety of stimuli, including heat shock, cytokine signalling, and virus infection ( Lang et al, 2010 ; Van Damme et al, 2010 ; Hoischen et al, 2018 ; Lang et al, 2019 ; Bernardi and Pandolfi, 2007 ; Maul et al, 1995 ). Infecting HSV-1 genomes are rapidly entrapped by PML-NBs upon nuclear entry ( Alandijany et al, 2018 ; Dembowski and Deluca, 2017 ), a host response that can lead to viral genome silencing as a component of the intrinsic antiviral immune response (discussed below) ( Alandijany et al, 2018 ; Everett et al, 2006 ; Everett et al, 2008a ; Glass and Everett, 2013 ; Cabral et al, 2018 ).…”