Atomic Force Microscopy Protocol for Measurement of Membrane Plasticity and Extracellular Interactions in Single Neurons in Epilepsy

Physiological interactions between extracellular matrix (ECM) proteins and membrane integrin receptors play a crucial role in neuroplasticity in the hippocampus, a key region involved in epilepsy. The atomic force microscopy (AFM) is a cutting-edge technique to study structural and functional measurements at nanometer resolution between the AFM probe and cell surface under liquid. AFM has been incrementally employed in living cells including the nervous system. AFM is a unique technique that directly measures functional information at a nanoscale resolution. In addition to its ability to acquire detailed 3D imaging, the AFM probe permits quantitative measurements on the structure and function of the intracellular components such as cytoskeleton, adhesion force and binding probability between membrane receptors and ligands coated in the AFM probe, as well as the cell stiffness. Here we describe an optimized AFM protocol and its application for analysis of membrane plasticity and mechanical dynamics of individual hippocampus neurons in mice with chronic epilepsy. The unbinding force and binding probability between ECM, fibronectin-coated AFM probe and membrane integrin were strikingly lower in dentate gyrus granule cells in epilepsy. Cell elasticity, which represents changes in cytoskeletal reorganization, was significantly increased in epilepsy. The fibronectin-integrin binding probability was prevented by anti-α5β1 integrin. Thus, AFM is a unique nanotechnique that allows progressive functional changes in neuronal membrane plasticity and mechanotransduction in epilepsy and related brain disorders

Hypoxia and Extracellular Matrix Proteins Influence Angiogenesis and Lymphangiogenesis in Mouse Embryoid Bodies

Regulatory mechanisms for angiogenesis are relatively well established compared to lymphangiogenesis. Few studies have shown that a combination of vascular endothelial growth factor VEGF-A/C with hypoxia or collagen matrix promotes lymphatic structures along with blood vessel development in mouse embryoid bodies (EB). In this study we tested the hypothesis that while hypoxia combined with prolonged VEGF-A/C treatment would induce early lymphangiogenesis in addition to angiogenesis in mouse EBs, under similar conditions specific extracellular matrix (ECM) proteins would promote lymphatic vessel-like structures over angiogenesis. EBs were subjected to four conditions and were maintained under normoxia and hypoxia (21% and 2.6% O2, respectively) with or without VEGF-A/C. Microarray analyses of normoxic and hypoxic EBs, and immunofluorescence data showed very low expression of early lymphatic endothelial cell (LEC) markers, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and prospero-related homeobox 1 (Prox1) at early time points. Double immunofluorescence using MECA-32 and Prox1/LYVE1 demonstrated that combined hypoxia and VEGF-A/C treatment promoted formation of blood vessel-like structures, whereas only Prox1+/LYVE1+ LECs were detected in EBs at E22.5. Furthermore, EBs were grown on laminin or collagen-I coated plates and were subjected to the four treatments as described above. Results revealed that LECs in EBs at E36.5 attached better to collagen-I, resulting in an organized network of lymphatic vessel-like structures as compared to EBs grown on laminin. However, blood vessel-like structures were less favored under these same conditions. Collectively, our data demonstrate that hypoxia combined with growth factors promotes angiogenesis, whereas combination of these conditions with specific ECM proteins favors lymphangiogenesis processes in mouse EBs

Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

The functional significance of the molecular swivel at the head-to-tail overlapping ends of contiguous tropomyosin (Tm) dimers in striated muscle is unknown. Contractile measurements were made in muscle fibers from transgenic (TG) mouse hearts that expressed a mutant α-Tm (Tm H276N ). We also reconstituted mouse cardiac troponin T (McTnT) N-terminal deletion mutants, McTnT 1–44 Δ and McTnT 45–74 Δ , into muscle fibers from Tm H276N . For controls, we used the wild-type (WT) McTnT because altered effects could be correlated with the mutant forms of McTnT. Tm H276N slowed crossbridge (XB) detachment rate ( g ) by 19%. McTnT 1–44 Δ attenuated Ca 2+ -activated maximal tension against Tm WT (36%) and Tm H276N (38%), but sped g only against Tm H276N by 35%. The rate of tension redevelopment decreased (17%) only in McTnT 1–44 Δ + Tm H276N fibers. McTnT 45–74 Δ attenuated tension (19%) and myofilament Ca 2+ sensitivity (pCa 50 =5.93 vs. 6.00 in the control fibers) against Tm H276N , but not against Tm WT background. Thus, altered XB cycling kinetics decreased the fraction of strongly bound XBs in McTnT 1–44 Δ + Tm H276N fibers, whereas diminished thin-filament cooperativity attenuated tension in McTnT 45–74 Δ + Tm H276N fibers. In summary, our study is the first to show that the interplay between the N terminus of cTnT and the overlapping ends of contiguous Tm effectuates different states of Tm on the actin filament. —Mamidi, R., Michael, J. J., Muthuchamy, M., Chandra, M. Interplay between the overlapping ends of tropomyosin and the N terminus of cardiac troponin T affects tropomyosin states on actin

Age-Related Alterations of Active Pumping Mechanisms in Rat Thoracic Duct

Objective: To evaluate the age-related changes in active pumping in thoracic duct (TD) from 24-month-old Fisher-344 rats comparing with TD pumping in 9-month rats. Methods: Lymphatic diameters, contraction amplitude and frequency, ejection fraction, and fractional pump flow were determined in isolated TD preparations. Western blot analyses were performed to evaluate relative levels of eNOS and iNOS in 9- and 24-month-old TD. Results: Stretch-dependent regulation was altered in aged TD especially at higher levels of pressure: the negative inotropy, negative chronotropy and diminished minute pumping (2- to 3-fold decrease) were observed. Physiological NO/imposed-flow-dependent inhibition was completely abolished in aged TD, yet NO-synthase blockade by l-NAME (10-4 M) increased pumping in a flow-independent manner. Western blot analyses indicated that the relative levels of eNOS were decreased ∼ 7-fold in the 24-month-old TD when compared with 9-month-old TD; whereas iNOS levels were increased ∼ 10-fold in 24-month-old TD. Conclusions: These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function