Identification of the domains within the N2A region of titin that regulate binding to actin.

Titin, the largest muscle protein, plays an important role in passive tension, sarcomeric integrity and cell signaling within the muscle. Recent work has also highlighted a role for titin in active muscle and the N2A region found in skeletal muscle titin and in some isoforms of cardiac titin has been linked to this function. The N2A region is a multi-domain region composed of four immunoglobulin domains (I80-I83) and a disordered region called the insertion sequence. Previously, our lab has shown that the N2A region binds F-actin in a calcium dependent manner, but it is not known which domains within this region are critical for this binding to occur. In this work, we have used co-sedimentation to demonstrate that only constructs containing the I80 domain are capable of binding F-actin. In addition, binding was only observed in constructs containing at least 3 immunoglobulin domains suggesting a length-dependence to binding. Finally, the calcium-dependence of N2A binding is lost when I83 is not present, consistent with the calcium stabilization that has been reported for this domain. Based on these results, we propose that I80 is critical for initiating binding to F-actin and that I83 is responsible for the calcium dependence.

Calcium increases titin N2A binding to F-actin and regulated thin filaments.

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A - F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A - actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A - actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin - actin binding occurs in skeletal muscle sarcomeres in vivo.

Engineering Remotely Triggered Liposomes to Target Triple Negative Breast Cancer.

Triple Negative Breast Cancer (TNBC) continues to present a challenge in the clinic, as there is still no approved targeted therapy. TNBC is the worst sub-type of breast cancer in terms of prognosis and exhibits a deficiency in estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. One possible option for the treatment of TNBC is chemotherapy. The issue with many chemotherapy drugs is that their effectiveness is diminished due to poor water solubility, and the method of administration directly or with a co-solvent intravenously can lead to an increase in toxicity. The issues of drug solubility can be avoided by using liposomes as a drug delivery carrier. Liposomes are engineered, biological nanoconstructs that possess the ability to encapsulate both hydrophobic and hydrophilic drugs and have been clinically approved to treat cancer. Specific targeting of cancer cell receptors through the use of ligands conjugated to the surface of drug-loaded liposomes could lessen damage to normal, healthy tissue. This study focuses on polyethylene glycol (PEG)-coated, folate conjugated, benzoporphyrin derivative (BPD)-loaded liposomes for treatment via photodynamic therapy (PDT). The folate receptor is over expressed on TNBC cells so these liposomes are targeted for greater uptake into cancer cells. PDT involves remotely irradiating light at 690 nm to trigger BPD, a hydrophobic photosensitive drug, to form reactive oxygen species that cause tumor cell death. BPD also displays a fluorescence signal when excited by light making it possible to image the fluorescence prior to PDT and for theranostics. In this study, free BPD, non-targeted and folate-targeted PEGylated BPD-loaded liposomes were introduced to a metastatic breast cancer cell line (MDA-MB-231) in vitro. The liposomes were reproducibly synthesized and characterized for size, polydispersity index (PDI), zeta potential, stability, and BPD release kinetics. Folate competition tests, fluorescence confocal imaging, and MTT assay were used to observe and quantify targeting effectiveness. The toxicity of BPD before and after PDT in monolayer and 3D in vitro cultures with TNBC cells was observed. This study may contribute to a novel nanoparticle-mediated approach to target TNBC using PDT.