Ramunas Stanciauskas

Gold nanoparticles continue to generate interest for use in several biomedical applications. Recently, researchers have been focusing on exploiting their dual diagnostic/therapeutic theranostic capabilities. Before clinical translation can occur, regulatory agencies will require a greater understanding of their biodistribution and safety profiles post administration. Previously, the real-time identification and tracking of gold nanoparticles in free-flowing vasculature had not been possible without extrinsic labels such as fluorophores. Here, we present a label-free imaging approach to examine gold nanoparticle (AuNP) activity within the vasculature by utilizing multiphoton intravital microscopy. This method employs a commercially available multiphoton microscopy system to visualize the intrinsic luminescent signal produced by a multiphoton absorption-induced luminescence effect observed in single gold nanoparticles at frame rates necessary for capturing real-time blood flow. This is the first demonstration of visualizing unlabeled gold nanoparticles in an unperturbed vascular environment with frame rates fast enough to achieve particle tracking. Nanoparticle blood concentration curves were also evaluated by the tracking of gold nanoparticle flow in vasculature and verified against known pre-injection concentrations. Half-lives of these gold nanoparticle injections ranged between 67 and 140 s. This label-free imaging approach could provide important structural and functional information in real time to aid in the development and effective analysis of new metallic nanoparticles for various clinical applications in an unperturbed environment, while providing further insight into their complex uptake and clearance pathways.

GRP78 conducts protein folding and quality control in the ER and shows elevated expression and cell surface translocation in advanced tumors. However, the underlying mechanisms enabling GRP78 to exert novel signaling functions at cell surface are just emerging. CD44 is a transmembrane protein and an important regulator of cancer metastasis, and isoform switch of CD44 through incorporating additional variable exons to the extracellular juxtamembrane region is frequently observed during cancer progression. Using super-resolution dual-color single-particle tracking, we report that GRP78 interacts with CD44v in plasma membrane nanodomains of breast cancer cells. We further show that targeting cell surface GRP78 by the antibodies can effectively reduce cell surface expression of CD44v and cell spreading of tamoxifen-resistant breast cancer cells. Our results uncover new functions of GRP78 as an interacting partner of CD44v and as a regulator of CD44v membrane homeostasis and cell spreading. This study also provides new insights into anti-CD44 therapy in tamoxifen-resistant breast cancer.

Mutagenic translesion DNA polymerase V (UmuD′₂C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′₂C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′₂C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecAE38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo. Escherichia coli upregulates more than 40 genes as part of the DNA damage-induced SOS regulon, many of which are involved in DNA repair and cell division. However, three DNA polymerases, pols V, II, and IV, are also induced to rescue replication forks blocked at persisting template lesions. Pol V (UmuD′₂C), encoded by the UV mutagenesis genes (umuDC), is primarily responsible for the increase in UV-induced chromosomal mutagenesis. However, pol V is catalytically inert. Interaction with a RecA nucleoprotein filament (RecA*) and ATP is required to convert pol V to an activated “mutasome” complex, pol V Mut = UmuD′₂C-RecA-ATP. Here, we show that pol V Mut deactivates dynamically during DNA synthesis, and statically in the absence of synthesis. Activated and deactivated states are governed by a conformational switch that repositions RecA relative to UmuC. Switching rates are more rapid at 37 than at 30°C, and do not require ATP hydrolysis. ATP (ATPγS) binding plays two required regulatory roles: 1) it allows binding of pol V Mut to primer-template DNA; 2) it triggers the RecA-UmuC conformational switch that activates pol V Mut.