NuclearActin

While the organisation of the cell nucleus is known to be a critical factor in many essential biological processes, the mechanisms that mediate and maintain this organisation are still not fully understood. Dr Maria Vartiainen tells us about her laboratory’s research into the role of actin in nuclear organisation

The cell nucleus is known to be a highly compartmentalised organelle, and this organisation plays a critical role in many essential biological processes, from gene expression to the maintenance of genomic integrity. The mechanisms that actually mediate and maintain this organisation are not fully understood however, an area that Dr Maria Vartiainen and her research group aim to investigate in the NuclearActin project. “The wider goal in our research is to look at how the cell nucleus is organised,” she outlines. The working hypothesis is that actin, a type of protein that is involved in many cellular processes, plays a critical role in this organisation, now Dr Vartiainen and her colleagues aim to investigate this further. “We’re using specific ways of manipulating actin in the nucleus, then we aim to identify what the actin is actually binding to in the nucleus, and thereby learn about how it contributes to nuclear organisation,” she says.

This represents a new area of research, as previously it had not been possible to manipulate nuclear actin levels specifically without affecting cytoplasmic actin, which is an important part of the cyto-skeleton and is central to many of the biological functions of cells. However, researchers have since been able to identify the mechanism by which actin is brought into the nucleus, opening up new avenues of investigation. “We have been able to identify the nuclear import mechanism for actin. This has enabled us to do this research,” says Dr Vartiainen. It is now possible to manipulate levels of actin in the nucleus while leaving cytoplasmic actin intact; this will enable researchers to look at a number of key questions around the biological significance of actin. “What happens if you reduce levels of nuclear actin? What happens to cellular functions? These are the underlying questions in our research,” says Dr Vartiainen.

Researchers are using the fruit fly drosophila melanogaster as a model system in this work, along with several mammalian cell lines, aiming to gain new insights into the molecular mechanisms by which actin operates in the nucleus. Nuclear actin itself has been linked to many processes in the nucleus, especially those involved in gene expression. “It has been linked to the regulation of transcription factor activity, to DNA damage response and to the function of the RNA polymerase for example,” says Dr Vartiainen. However, little is known about the role of actin in relation to the function of the RNA polymerase and many other important biological processes. “That’s why we want to understand the binding partners for nuclear actin for example. We can then build a deeper understanding of how it actually affects these important nuclear complexes. This essentially applies to all nuclear actin functions,” explains Dr Vartiainen.

A key step is of course to first identify the proteins that bind to nuclear actin. Dr Vartiainen and her colleagues are breaking new ground in this respect with the development of sophisticated new methods. “We have developed novel microscopic methods to identify the binding partners, that’s a new technical development. We are also creating new models to study nuclear actin,” she outlines. A novel multi read-out fluorescence microscopy screen will be implemented within the project, from which researchers can identify the binding partners of nuclear actin and investigate them in greater depth than previously possible. “The fluorescence microscopy screen itself has multiple readouts, so in addition to identifying the novel binding partners, we can also study the effect of these partners on transcription factor activity,” continues Dr Vartiainen.

The project is using several different techniques to analyse these proteins, including chromatin immunoprecipitation, sequencing and transcriptomics. Researchers aim to gain

GFP-actin expressing cells in control (left) and Exportin-6 (right) depleted conditions, which block nuclear export of actin, and thus accumulates actin in the nucleus.

a deeper understanding of the biological significance of nuclear actin through this work. “We’re looking at fundamental processes around gene expression,” says Dr Vartiainen. While research in the NuclearActin project is largely fundamental in nature, dysregulation of actin has been linked to failures in cell function, something which Dr Vartiainen and her colleagues are investigating in

the laboratory. “We recently collaborated on a paper with researchers in the US, where we showed that in certain laminopathies there is a defect in nuclear actin polymerisation. Laminopathies are a group of diseases caused by mutations in the nuclear envelope proteins, and they often lead to myocardial and cardiac dysfunction,” she outlines.

A number of questions remain around the role of actin in laminopathies, and Dr Vartiainen plans to pursue further research in this area in future, outside the current scope of the NuclearActin project. Within the project itself, researchers will continue to investigate the biological significance of nuclear actin. “We want to identify which genes depend on actin for their expression – we’ve already gained a lot of data on that, and we’re currently checking through it. We hope to identify novel nuclear

functions that depend on actin, through these novel binding partners,” says Dr Vartiainen. Researchers now plan to publish some of the initial results from the project. “We need to publish the results from the fruit fly models, and we also have the preliminary analysis of the nuclear actin interactions,” continues Dr Vartiainen. “This project has also resulted in many novel lines of research that we are going to continue to pursue in the laboratory.”

Actin as the Master Organizer of Nuclear Structure and Function (NuclearActin)

The cell nucleus is a highly compartmentalized organelle, and its organization has critical for many essential processes, from gene expression to maintenance of genomic integrity. NuclearActin project studies the role of actin in nuclear organization, by using a combination of genomic and imaging techniques.

Main funding ERC Starting grant, other funding of the lab: Academy of Finland, Sigrid Juselius foundation, Finnish Cancer Foundation and Biocentrum Helsinki.

Project Coordinator, Institute of Biotechnology University of Helsinki PO BOX 56 00014 Helsinki Finland T: + 35 8919 159419 E: maria.vartiainen@helsinki.fi W: http://www.biocenter.helsinki.fi/bi/ vartiainen/

Dopie J, Skarp KP, Rajakylä EK, Tanhuanpää K, Vartiainen MK. Active maintenance of nuclear actin by Importin 9 supports Transcription. PNAS 2012 109(9):E544-52

Dr Maria Vartiainen

Dr Maria Vartiainen obtained her PhD a in 2002 from University of Helsinki, and then worked as a post-doc with Richard Treisman at LRI-CRUK in London. Since 2007, Maria has been a group leader at the Institute of Biotechnology in Helsinki, acting also as the scientific leader of the Light Microscopy Unit.