Temporal Patterns of Angular Displacement of Endosomes: Insights into Motor Protein Exchange Dynamics.

The material transport system, facilitated by motor proteins, plays a vital role in maintaining a non-equilibrium cellular state. However, understanding the temporal coordination of motor protein activity requires an advanced imaging technique capable of measuring 3D angular displacement in real-time. In this study, a Fourier transform-based plasmonic dark-field microscope has been developed using anisotropic nanoparticles, enabling the prolonged and simultaneous observation of endosomal lateral and rotational motion. A sequence of discontinuous 3D angular displacements has been observed during the pause and run phases of transport. Notably, a serially correlated temporal pattern in the intermittent rotational events has been demonstrated during the tug-of-war mechanism, indicating Markovian switching between the exploitational and explorational modes of motor protein exchange prior to resuming movement. Alterations in transition frequency and the exploitation-to-exploration ratio upon dynein inhibitor treatment highlight the relationship between disrupted motor coordination and reduced endosomal transport efficiency. Collectively, these results suggest the importance of orchestrated temporal motor protein patterns for efficient cellular transport.

Mid-old cells are a potential target for anti-aging interventions in the elderly.

The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.

Single-Molecule Rotation for EGFR Conformational Dynamics in Live Cells.

Monitoring the dynamics of proteins in live cells on appropriate spatiotemporal scales may provide key information regarding long-standing questions in molecular and cellular regulatory mechanisms. However, tools capable of imaging the conformational changes over time have been elusive. Here, we present a single-molecule stroboscopic imaging probes by developing gyroscopic plasmonic nanoparticles, allowing for replication of protein-protein interactions and the conformational dynamics based on rotational and lateral velocities. This study fundamentally monitors the rotational motion of a membrane protein, epidermal growth factor receptor (EGFR), to decipher undiscovered structural dynamics in live cells without any molecular perturbations. This method offers a strategy to visualize assemblies and conformational changes, and provides unique insights into the mechanism underlying the molecular dynamics for receptors.

Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles.

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (µMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the µMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.

Production and targeting of monovalent quantum dots.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

Sensitive and selective plasmon ruler nanosensors for monitoring the apoptotic drug response in leukemia.

Caspases are proteases involved in cell death, where caspase-3 is the chief executioner that produces an irreversible cutting event in downstream protein substrates and whose activity is desired in the management of cancer. To determine such activity in clinically relevant samples with high signal-to-noise, plasmon rulers are ideal because they are sensitively affected by their interparticle separation without ambiguity from photobleaching or blinking effects. A plasmon ruler is a noble metal nanoparticle pair, tethered in close proximity to one another via a biomolecule, that acts through dipole-dipole interactions and results in the light scattering to increase exponentially. In contrast, a sharp decrease in intensity is observed when the pair is confronted by a large interparticle distance. To align the mechanism of protease activity with building a sensor that can report a binary signal in the presence or absence of caspase-3, we present a caspase-3 selective plasmon ruler (C3SPR) composed of a pair of Zn0.4Fe2.6O4@SiO2@Au core-shell nanoparticles connected by a caspase-3 cleavage sequence. The dielectric core (Zn0.4Fe2.6O4@SiO2)-shell (Au) geometry provided a brighter scattering intensity versus solid Au nanoparticles, and the magnetic core additionally acted as a purification handle during the plasmon ruler assembly. By monitoring the decrease in light scattering intensity per plasmon ruler, we detected caspase-3 activity at single molecule resolution across a broad dynamic range. This was observed to be as low as 100 fM of recombinant material or 10 ng of total protein from cellular lysate. By thorough analyses of single molecule trajectories, we show caspase-3 activation in a drug-treated chronic myeloid leukemia (K562) cancer system as early as 4 and 8 h with greater sensitivity (2- and 4-fold, respectively) than conventional reagents. This study provides future implications for monitoring caspase-3 as a biomarker and efficacy of drugs.

Poly(ethylene glycol)- and carboxylate-functionalized gold nanoparticles using polymer linkages: single-step synthesis, high stability, and plasmonic detection of proteins.

Gold nanoparticles with suitable surface functionalities have been widely used as a versatile nanobioplatform. However, functionalized gold nanoparticles using thiol-terminated ligands have a tendency to aggregate, particularly in many enzymatic reaction buffers containing biological thiols, because of ligand exchange reactions. In the present study, we developed a one-step synthesis of poly(ethylene glycol) (PEG)ylated gold nanoparticles using poly(dimethylaminoethyl methacrylate) (PDMAEMA) in PEG as a polyol solvent. Because of the chelate effect of polymeric functionalities on the gold surface, the resulting PEGylated gold nanoparticles (Au@P-PEG) are very stable under the extreme conditions at which the thiol-monolayer-protected gold nanoparticles are easily coagulated. Using the solvent mixture of PEG and ethylene glycol (EG) and subsequent hydrolysis, gold nanoparticles bearing mixed functionalities of PEG and carboxylate are generated. The resulting particles exhibit selective adsorption of positively charged chymotrypsin (ChT) without nonselective adsorption of bovine serum albumin (BSA). The present nanoparticle system has many advantages, including high stability, simple one-step synthesis, biocompatibility, and excellent binding specificity; thus, this system can be used as a versatile platform for potential bio-related applications, such as separation, sensing, imaging, and assays.

Formation of targeted monovalent quantum dots by steric exclusion.

Precise control over interfacial chemistry between nanoparticles and other materials remains a major challenge that limits broad application of nanotechnology in biology. To address this challenge, we used 'steric exclusion' to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping each QD with a functionalized oligonucleotide. We demonstrated the utility of these QDs as modular and nonperturbing imaging probes by tracking individual Notch receptors on live cells.