Summary Fluid shear stress (FSS) from blood flow acting on the endothelium critically regulates vascular morphogenesis, blood pressure and atherosclerosis [1]. FSS applied to endothelial cells (EC) triggers signaling events including opening of ion channels, activation of signaling pathways and changes in gene expression. Elucidating how ECs sense flow important for understanding both normal vascular function and disease. EC responses to FSS are mediated in part by a junctional mechanosensory complex consisting of VE-cadherin, PECAM-1, and VEGFR2 [2]. Previous work suggested that flow increases force on PECAM-1, which initiates signaling [2–4]. Deletion of PECAM-1 blocks responses to flow in vitro and flow-dependent vascular remodeling in vivo [2, 5]. To understand this process, we developed and validated FRET-based tension sensors for VE-cadherin and PECAM-1 using our previously developed FRET tension biosensor [6]. FRET measurements showed that in static culture, VE-cadherin in cell-cell junctions bears significant myosin-dependent tension, whereas there was no detectable tension on VE-cadherin outside of junctions. Onset of shear stress triggered a rapid (<30 sec) decrease in tension across VE-cadherin, which paralleled a decrease in total cell-cell junctional tension. Flow triggered a simultaneous increase in tension across junctional PECAM-1, while non-junctional PECAM-1 was unaffected. Tension on PECAM-1 was mediated by flow-stimulated association with vimentin. These data confirm the prediction that shear increases force on PECAM-1. However, they also argue against the current model of passive transfer of force through the cytoskeleton to the junctions [7], showing instead that flow triggers cytoskeletal remodeling, which alters forces across the junctional receptors.

Nanoparticle size, surface charge and material composition are known to affect the uptake of nanoparticles by cells. However, whether nanoparticle shape affects transport across various barriers inside the cell remains unclear. Here we used pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers at different rates, ultimately defining the site of drug release. We measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from the endosomal system and had access to the nucleus via the nuclear pore complex. Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion. Improving nuclear access, for example with a nuclear localization signal, resulted in more doxorubicin release inside the nucleus and correlated with greater cytotoxicity. Our results therefore demonstrate that drug delivery across the major cellular barrier, the nuclear envelope, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.

No methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here, we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase nucleus of live cells. In the pCF method, we cross-correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of ≈300 ms, suggesting intrinsic localized change in chromatin structure. This is a unique in vivo demonstration of the intricate chromatin network showing channel directed diffusion of an inert molecule with high spatial and temporal resolution.chromatin organization | fluctuation spectroscopy | nucleus structure N uclear architecture is fundamental to the manner in which molecules traverse the nucleus (1). The cell nucleus is a functionally and spatially structured organelle (2) in which diffusion is the mode of motion for inert molecules (3-5). The diffusion of molecules within the nucleus is obstructed by the steric constraints imposed by structural components, such as chromatin (6, 7). Proposed models for 3D arrangement of chromatin vary from defined regions of chromatin compartmentalisation (8) to intermingled chromatin fibers and loops (9). The requirement for biologically significant molecules to reach different destinations within the cell nucleus raises the question, how is the diffusive route directed? Given that diffusion cannot be regulated because it is essentially a default mechanism of motion (10), it has been postulated that structural features of the nucleus must impart retention at particular sites and control flux of movement between compartments (1, 11).Insights into intranuclear trafficking are predominantly derived from measurement of the accessibility of the nuclear landscape and the effect it has on diffusion of biologically active and inert molecules. Current approaches commonly used for such investigations are fluorescence recovery after photo bleaching (FRAP) (12, 13), single particle tracking (SPT) (14), and fluorescence correlation spectroscopy (FCS) (15, 16). FRAP, however, is invasive because it requires high illumination power on the sample to induce photobleaching, and SPT requires the observation of isolated particles for a long time, which yields poor statistics. FCS, in contrast, provides information at the single molecule level with good statistics by averaging the behavior of many molecules (10), and this technique ...