Research Projects

1. The nuclear envelope in cell surface to nucleus signaling

2. Imaging and analysis of cell signaling in live neuronal cells

3. Nuclear envelope proteins in mitosis and maintenance of genomic stability

 

The nuclear envelope acts as a signaling node to provide enhanced diversification in cell surface to nucleus signaling in eukaryotic cells, a phenomenon which may be especially important in the highly polarized and elongated neuronal cell.

Nuclear envelope
Nuclear envelope
 

Fig.1. Organization of the nuclear envelope (modified from Stewart et al., 2007)

Background


The nuclear envelope (NE) that surrounds the genetic material in eukaryotic cells (Fig.1) consists of two concentric membranes, the inner nuclear membrane (INM) and outer nuclear membranes (ONM), the nuclear lamina, the nuclear pores and nuclear pore complexes (NPCs). The NPCs are responsible for import and export of proteins and RNA molecules in and out of the nucleus. In addition, a whole new concept in cell signaling bypassing the nuclear pores is provided by the recently discovered LINC complexes (Fig.1), which are built up by specific proteins in the inner and outer nuclear membranes. These direct links across the NE that connect the cytoskeleton with the nuclear interior are believed to mediate mechanical signals from the cell surface to the nuclear interior. The LINC complexes also play important roles in migration of nuclei taking place, e.g. when neuronal cells divide in the developing mammalian brain as well as in muscles for specific positioning of nuclei at neuromuscular junctions.

 

Human diseases such as cancer have been tied to proteins of the NPC. Nuclear lamins and proteins of the INM have been genetically linked to a diverse group of diseases collectively termed “laminopathies”, which include muscular and lipid dystrophies, neurological disorders and progeria (premature aging). As a result of these discoveries it has now been realized that proteins of the NE orchestrate a much larger repertoire of functions than previously realized, both in cell signaling, chromatin organization and in the mitotic machinery. The INM has been estimated to contain nearly a hundred unique transmembrane proteins, of which the large majority is still not characterized.

 

Project 1. The nuclear envelope in cell surface to nucleus signaling


Signals from the cell surface may reach the nuclear interior via import of signaling proteins through the nuclear pores (Arabi et al., 2003; Hallberg et al., 1993; Onischenko et al., 2005). However, a whole new concept is provided by the recently discovered LINC complexes, which can mediate direct mechanical transduction of signals from the cytoskeleton to the nuclear interior bypassing the nuclear pores. On the nuclear side the LINC complex interacts with transmembrane proteins of the INM and the nuclear lamina, which may in turn respond by directly or indirectly change chromatin organization and gene activity and or sequester transcription factors.

 

We are focusing on investigating the function(s) of specific networks of interactions between proteins in the NE and their role in cellular signaling and chromatin organization. As an example, we have recently identified and characterized a novel transmembrane protein from the INM, Samp1 (Buch et al., 2009). Samp1 is conserved from fission yeast to man and contains four conserved Cys-X-X-Cys motifs suggesting that it is able to form two Zinc fingers. Furthermore, Samp1 is essential for connecting the centrosome close to the NE (Buch et al., 2009) and our more recent data strongly suggest that it is connected to the LINC complex (Gudise et al., 2011) and thus may play an important role in cell surface to nucleus signaling.

 

Project 2. Imaging and analysis of cell signaling in live neuronal cells


We have extensive expertise in imaging and analysis in live cells, including FRAP, FLIP and FRET (Arabi et al., 2003; Buch et al., 2009; Daigle et al., 2001; Eriksson et al., 2004; Figueroa et al., 2011; Ivanova et al., 2016; Le Rouzic et al., 2002; Onischenko et al., 2005; Östlund et al., 1999). The extended tubular shape of neurites addresses particular questions regarding diffusion-mediated signaling in neuronal cells, which may to a large extent rely on motor driven propagation of signals between the cell surface and the nucleus. For example, we have designed a series of anchored FRET (Fluorescences Resonance Energy Transfer) sensor molecules that enable monitoring of local activation of different caspases in neurites and soma of degenerating neuronal cells (Figueroa et al., 2011; Ivanova et al., 2016). We will now use cells expressing these FRET sensors as a model to study mechanisms behind neurodegenerative diseases such as Alzheimers disease (AD). One of our future aims is to develop techniques to image and analyze motor driven signaling in live neuronal cells
 

Project 3. Nuclear envelope proteins in mitosis and maintenance of genomic stability

 

Normally, an intricate system of spindle assembly factors and checkpoints assuring correct assembly and maturation of the mitotic spindle is essential for symmetric chromosome segregation and thus maintenance of genomic stability. Defects in the mitotic performance are the main cause of aneuploidy and cancer. Neuroblastoma is the third commonest form of cancer in children and is believed to arise from neuronal progenitor cells during development of the sympathetic nervous system. Genetic instability, manifested as amplification of the MYCN gene, is associated with the aggressive form of the disease.

We use differentiating human neuronal progenitor stem cells as a model system for the development of neuroblastoma in human children. We are focusing on a transmembrane protein of the inner nuclear membrane, Samp1 (Spindle associated membrane proten 1), because it is implicated to play a role in the mitotic machinery and because it specifically concentrates along kinetochore microtubule in mitosis (Fig. 2) (Buch et al., 2009). Recent results show that Samp1 is essential for correct chromosome segregation and binds directly to RanGTPase (Vijayaraghavan et al., 2016), which is a key regulator of mitotic spindle assembly.
 
 

 

Fig. 2. Samp1, an integral membrane protein of the INM in mitotic spindle membranes. Samp1 (green) concentrates in membranes along kinetochore microtubules of HeLa cells projecting from spindle poles (magenta) towards metaphase chromosomes (red). From (Buch et al., 2009b)
 

 

Selected publications
 
Arabi, A., C. Rustum, E. Hallberg, and A.P. Wright. 2003. Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels. J Cell Sci. 116:1707-1717.
 
Buch, C., R. Lindberg, R. Figueroa, S. Gudise, E. Onischenko, and E. Hallberg. 2009b. An integral protein of the inner nuclear membrane localizes to the mitotic spindle in mammalian cells. J Cell Sci. 122:2100-7.
 
Daigle, N., J. Beaudouin, L. Hartnell, G. Imreh, E. Hallberg, J. Lippincott-Schwartz, and J. Ellenberg. 2001. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J Cell Biol. 154:71-84.
 
Eriksson, C., C. Rustum, and E. Hallberg. 2004. Dynamic properties of nuclear pore complex proteins in gp210 deficient cells. FEBS Lett. 572:261-5.
 

Figueroa, R.A., V. Ramberg, T. Gatsinzi, M. Samuelsson, M. Zhang, K. Iverfeldt, and E. Hallberg. 2011. Anchored FRET sensors detect local caspase activation prior to neuronal degeneration. Molecular neurodegeneration. 6:35.

Gudise, S., R.A. Figueroa, R. Lindberg, V. Larsson, and E. Hallberg. 2011. Samp1 is functionally associated with the LINC complex and A-type lamina networks. J Cell Sci. 124:2077-2085.

 
Hallberg, E., R.W. Wozniak, and G. Blobel. 1993. An Integral Membrane Protein of the Pore Membrane Domain of the Nuclear Envelope Contains a Nucleoporin-Like Region. J. Cell Biol. 122:513-521.
 

Ivanova, E.V., R.A. Figueroa, T. Gatsinzi, E. Hallberg, and K. Iverfeldt. 2016. Anchoring of FRET Sensors-A Requirement for Spatiotemporal Resolution. Sensors (Basel). 16.

Le Rouzic, E., A. Mousnier, C. Rustum, F. Stutz, E. Hallberg, C. Dargemont, and S. Benichou. 2002. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1. J Biol Chem. 277:45091-8.
 
Onischenko, E.A., N.V. Gubanova, E.V. Kiseleva, and E. Hallberg. 2005. Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos. Mol Biol Cell. 16:5152-62.
 

Vijayaraghavan, B., M.H. Jafferali, R.A. Figueroa, and E. Hallberg. 2016. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane. Nucleus:0.

Östlund, C., J. Ellenberg, E. Hallberg, J. Lippincott-Schwartz, and H.J. Worman. 1999. Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein. J. Cell Sci. 112:1709-1719.