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Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived ‘signaling-capable’ CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

Fluorescence correlation spectroscopy (FCS) is an optical technique in which the fluctuations in fluorescence intensity are quantified. The time correlation function gives insight into the dynamics of the molecule in its environment: typically the diffusion coefficient in a dilute solution is measured, but the technique has been expanded to uses in more complex environments, including living cells. In these environments photobleaching and dye-dissociation can substantially introduce artifacts in the FCS data. We present a technique to correct for the artifacts introduced by photobleaching and study dye dissociation in DNA solutions.

Integrins are transmembrane adhesion receptors that partner with extracellular ligands and cytoskeletal proteins to form multi-protein adhesive complexes known as focal adhesions. These complexes span across large, micron-sized membrane domains and provide a link between the cell and its environment. Additionally, they serve as cell signaling hubs, regulating cell growth and motility by relaying mechanical and biochemical signals throughout the cell. Focal adhesion functionality is hypothesized to be sensitive to cell membrane composition, driven at least in part by the tendency for the plasma membrane to separate into ordered and disordered domains resembling liquid phases observed in model membranes.

DNA looping is a vital regulatory process in the cell to maintain proper function and viability. In protein-mediated looping, the DNA's sequence dictates both protein binding sites and the local mechanical properties of the DNA. These mechanical properties, notably its elasticity and intrinsic curvature, govern the ease at which loops can form. To probe the effects of sequence dependence on elasticity and the loop formation process, single molecule experiments were performed on short segments (∼150bp) of DNA with varying amounts of AT and GC content between the protein-binding operators. Tethered particle motion (TPM) microscopy was employed to observe protein-mediated DNA loop formation in this system, and an analytical model of the loop formation process was used to calculate the elasticity of the DNA from the observed loop formation rates. For comparison, axial constant-force optical tweezers were used to directly stretch the same DNA molecules mechanically to determine their persistence length as a measure for their elasticity. Our results indicate that the intraoperator sequence has a larger effect on elasticity in the loop formation experiments than in the stretching experiments, which we attribute to different elasticity regimes when the DNA is strongly bent as in a DNA loop, compared to the thermally induced small curvature fluctuations in stretched DNA.

We have performed de Haas-van Alphen measurements of the Fermi surface of alpha-uranium single crystals at ambient pressure within the alpha-3 charge density wave (CDW) state from 0.020 K - 10 K and magnetic fields to 35 T using torque magnetometry. The angular dependence of the resulting frequencies is described. Effective masses were measured and the Dingle temperature was determined to be 0.74 K +/- 0.04 K. The observation of quantum oscillations within the alpha-3 CDW state gives new insight into the effect of the charge density waves on the Fermi surface. In addition we observed no signature of superconductivity in either transport or magnetization down to 0.020 K indicating the possibility of a pressure-induced quantum critical point that separates the superconducting dome from the normal CDW phase., Comment: 11 pages, 4 figures, 3 tables