7th Annual Symposium
Physics of Cancer
October 4-6, 2016
|PoC - Physics of Cancer - Annual Symposium|
LINC-ing the nucleus, the cytoskeleton and cancer
Contact: | Website
Cancer is now the leading cause of death in economically developed countries. The presence of secondary tumors (also called metastases) are a poor prognostic factor, and the cause of 90% of cancer deaths. A promising strategy to reduce mortality is to target the mechanisms by which cells leave the primary tumour and disseminate through the body. To migrate through tissues, cells have to overcome the mechanical resistance of their large and rigid nucleus, which I (and others) have shown to be a limiting factor during 3-D migration. Considerable force needs to be applied by the cytoskeleton to the nucleus, yet many aspects of mechanical linkage and force transmission from the cytoskeleton to the nucleus are still poorly understood. Many nuclear envelope proteins, including nesprins, which mechanically link the actin cytoskeleton to the nuclear membrane have been implicated in cancer. The cytoskeletal actors that mediate force exertion on the nucleus to translocate it through narrow constrictions have yet to be fully identified. However, non-muscle myosin IIB is necessary for cell migration through constrictions in breast cancer and glioma cells, strongly implying that actomyosin contractility is the major contributor. Furthermore, actin-associated proteins which bind nesprins such as FHOD1 are necessary for nuclear movement and fascin is required for nuclear translocation through narrow constrictions.
My hypothesis is that nesprin expression is altered in migrating cancer cells, allowing more efficient transmission of forces to the nucleus and therefore more efficient 3D migration. To verify this hypothesis, I am investigating the nesprin isoform composition of metastatic cells, verifying the ability of these cells to migrate through narrow 3-D constrictions more efficiently, and rebuilding and studying the nesprin-actin physical connection in vitro.
Nesprin isoform composition of metastatic cells.
The nesprin family is comprised of four genes that encode many different isoforms. Giant nesprin isoforms of nesprins-1 and 2 (~1mDa and 800 kDa, resp.) anchor into the nuclear envelope through their KASH domain by binding SUN proteins (themselves anchored into the nuclear lamina) and they bind actin fibers directly through calponin homology (CH) domains. These connections are crucial for many processes that involve force transmission to the nucleus. SYNE1 and 2, which encode nesprin-1 and 2, are candidate cancer genes, and an antibody for the actin-binding domain of nesprin 2 showed increased expression in tumours, but the expression of nesprins in metastatic cells has yet to be fully investigated.
Using an antibody that recognizes the KASH domain of nesprins-1 and 2, I used western blot to identify the expression levels of two patient metastatic cell lines and MCF-10A cell (healthy breast cell line). The highest molecular weight bands detected (presumably the giant isoforms) were clearly labelled for both metastatic cell lines, but only faintly detectable for nesprin-2 for the control cells. The control cells predominantly expressed lower molecular weight isoforms.
Ability of metastatic cells to migrate through narrow constrictions.
I compared the migration ability of these cells in devices that I developed. By comparing the migration time of the nucleus through 3 µm constrictions to the time required to pass through a 15 µm control channel, I obtain the normalized migration time indicative of the barrier that the nucleus represents during migration. The two metastatic cell lines had shorter normalized migration times than the control cells, indicating that they passed their nucleus through constrictions more easily than control cells, despite equivalent or higher lamin A/C levels (and consequently increased nuclear stiffness). Thus, the metastatic cells appear to have an increased ability to deform their nucleus through narrow constrictions compared to the control cells, independently of stiffness.
Rebuilding actin-nucleus interactions in vitro.
I isolated nuclei from fibroblasts using a hypotonic buffer and a shearing protocol. I showed that nesprins at the nuclear membrane are recognized using antibodies for the calponin homology domain, indicating that these domains are intact following the isolation procedure. In vitro reconstitution of actin filaments in the presence of isolated nuclei shows that filaments coat the surface of the nuclei. Using an antibody that masks the actin-binding site of nesprin significantly reduces this interaction, implying that we are specifically observing actin binding to nesprins.
Future experiments to characterize the biophysical properties of the nesprin-actin interaction are planned. These include measuring the mobility and recruitment of nesprins in presence and absence of actin (FRAP experiments, actin shell peeling, deposition of nuclei on actin patches). I will also characterize the amount of force that can be transferred to the nesprin-actin connection using optical tweezers, and determine whether nesprins bind more strongly to actin architectures that resemble peri-nuclear actin, and whether nesprins can induce actin organization.
I demonstrate here that metastatic patient cells are able to deform their nucleus more efficiently to migrate in 3-D small spaces and show increased levels of the giant isoforms of nesprins-1 and 2, indicating an increased ability to mechanically link their nucleus to the actin cytoskeleton for force exertion. By rebuilding the interactions between the nucleus and reconstituted actin filaments in vitro, I will characterize the biophysical interactions between nesprins and actin.