Why a Particle Physicist is Interested in DNA Branch Migration

We describe an explicitly discrete model of the process of DNA branch migration. The model matches the existing data well, but we find that branch migration along long strands of DNA (N \simge 40~bp) is also well modeled by continuum diffusion. The discrete model is still useful for guiding future experiments.Comment: Talk presented at LATTICE96(theoretical developments); 3 pages, TeXsis w/ LAT96.txs (available from ftp://lifshitz.ph.utexas.edu/texsis/styles/LAT96.txs and will be a part of the next Elsevier.txs) and TXSdcol.te

Biomimetic and Biophysical Approach to Profile Metastatic Cancer Cell Migration

Honors Research ScholarshipCancer metastasis is a complex process by which cells in a primary tumor acquire an aggressive phenotype, and travel to distant, secondary sites in the body. One aspect of cancer metastasis is cell migration toward the vascular system, called invasion. Multiple modalities of single cell invasion exist, including amoeboid migration and mesenchymal migration. Amoeboid migration is less well understood, and in particular, the forces involved in amoeboid migration have yet to be fully elucidated at a subcellular scale. Cellular traction force microscopy, or CTFM, is one method used to probe migration forces. However, this approach is largely limited to two dimensions, and is limited by the size of the pillars on the substrate. To address these limitations, we developed a system using microfluidics and DNA origami capable of real-time force measurement of cell migration on a subcellular scale with a 10 pN resolution. Microfluidic devices were made using soft lithography and replica molding in our laboratory. DNA origami were made using protocols developed by Michael Hudoba and Dr. Carlos Castro in the Nanoengineering and Biodesign Laboratory. The devices were imaged using TIRF microscopy to study dwell times of the sensors in the open and closed states, and the devices were analyzed with an AFM to determine that they are best suited for measuring shear forces. Further, the presence of streptavidin protein was found to have a significant effect on DOFS binding with a p-value less than 0.05. DOFS concentrations around 1 nM were found to provide the most coverage while minimizing structure aggregation. Thus, our microfluidic devices are able to be functionalized with DNA origami force sensors with a high degree of attachment. This platform is thus capable of measuring cell migration and adhesion forces, and future work should harness this system to create 3D maps of cell migration to gain insight into invasion.Institute for Materials ResearchSecond-Year Transformational Experience Program (STEP)A one-year embargo was granted for this item.Academic Major: Biomedical Engineerin

Two-State Migration of DNA in a structured Microchannel

DNA migration in topologically structured microchannels with periodic cavities is investigated experimentally and with Brownian dynamics simulations of a simple bead-spring model. The results are in very good agreement with one another. In particular, the experimentally observed migration order of Lambda- and T2-DNA molecules is reproduced by the simulations. The simulation data indicate that the mobility may depend on the chain length in a nonmonotonic way at high electric fields. This is found to be the signature of a nonequilibrium phase transition between two different migration states, a slow one and a fast one, which can also be observed experimentally under appropriate conditions.Comment: Revised edition corresponding to the comments by the referees, submitted to Physical Review

Fluctuation-Facilitated Charge Migration along DNA

We propose a model Hamiltonian for charge transfer along the DNA double helix with temperature driven fluctuations in the base pair positions acting as the rate limiting factor for charge transfer between neighboring base pairs. We compare the predictions of the model with the recent work of J.K. Barton and A.H. Zewail (Proc.Natl.Acad.Sci.USA, {\bf 96}, 6014 (1999)) on the unusual two-stage charge transfer of DNA.Comment: 4 pages, 2 figure

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation

Thermal diffusion by Brownian motion induced fluid stress

The Ludwig-Soret effect, the migration of a species due to a temperature gradient, has been extensively studied without a complete picture of its cause emerging. Here we investigate the dynamics of DNA and spherical particles sub jected to a thermal gradient using a combination of Brownian dynamics and the lattice Boltzmann method. We observe that the DNA molecules will migrate to colder regions of the channel, an observation also made in the experiments of Duhr, et al[1]. In fact, the thermal diffusion coefficient found agrees quantitatively with the experimental value. We also observe that the thermal diffusion coefficient decreases as the radius of the studied spherical particles increases. Furthermore, we observe that the thermal fluctuations-fluid momentum flux coupling induces a gradient in the stress which leads to thermal migration in both systems.Comment: 6 pages, 5 figue

Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe

A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transfered and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involve

Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells

As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-κB (NF-κB) signaling and a tumor necrosis factor-α (TNFα)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics