Dongyoung Kim

3D Single Molecule Tracking with Multifocal Plane Microscopy Reveals Rapid Intercellular Transferrin Transport at Epithelial Cell Barriers

The study of intracellular transport pathways at epithelial cell barriers that line diverse tissue sites is fundamental to understanding tissue homeostasis. A major impediment to investigating such processes at the subcellular level has been the lack of imaging approaches that support fast three-dimensional (3D) tracking of cellular dynamics in thick cellular specimens. Here, we report significant advances in multifocal plane microscopy and demonstrate 3D single molecule tracking of rapid protein dynamics in a 10 micron thick live epithelial cell monolayer. We have investigated the transferrin receptor (TfR) pathway, which is not only essential for iron delivery but is also of importance for targeted drug delivery across cellular barriers at specific body sites, such as the brain that is impermeable to blood-borne substances. Using multifocal plane microscopy, we have discovered a cellular process of intercellular transfer involving rapid exchange of Tf molecules between two adjacent cells in the monolayer. Furthermore, 3D tracking of Tf molecules at the lateral plasma membrane has led to the identification of different modes of endocytosis and exocytosis, which exhibit distinct temporal and intracellular spatial trajectories. These results reveal the complexity of the 3D trafficking pathways in epithelial cell barriers. The methods and approaches reported here can enable the study of fast 3D cellular dynamics in other cell systems and models, and underscore the importance of developing advanced imaging technologies to study such processes.

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.

Antibody targeting of HER2/HER3 signaling overcomes heregulin‐induced resistance to PI3K inhibition in prostate cancer

Dysregulated expression and/or mutations of the various components of the phosphoinositide 3‐kinase (PI3K)/Akt pathway occur with high frequency in prostate cancer and are associated with the development and progression of castration resistant tumors. However, small molecule kinase inhibitors that target this signaling pathway have limited efficacy in inhibiting tumor growth, primarily due to compensatory survival signals through receptor tyrosine kinases (RTKs). Although members of the epidermal growth factor receptor (EGFR), or HER, family of RTKs are strongly implicated in the development and progression of prostate cancer, targeting individual members of this family such as EGFR or HER2 has resulted in limited success in clinical trials. Multiple studies indicate a critical role for HER3 in the development of resistance against both HER‐targeted therapies and PI3K/Akt pathway inhibitors. In this study, we found that the growth inhibitory effect of GDC‐0941, a class I PI3K inhibitor, is markedly reduced in the presence of heregulin. Interestingly, this effect is more pronounced in cells lacking phosphatase and tensin homolog function. Heregulin‐mediated resistance to GDC‐0941 is associated with reactivation of Akt downstream of HER3 phosphorylation. Importantly, combined blockade of HER2 and HER3 signaling by an anti‐HER2/HER3 bispecific antibody or a mixture of anti‐HER2 and anti‐HER3 antibodies restores sensitivity to GDC‐0941 in heregulin‐treated androgen‐dependent and ‐independent prostate cancer cells. These studies indicate that the combination of PI3K inhibitors with HER2/HER3 targeting antibodies may constitute a promising therapeutic strategy for prostate cancer.

The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells

The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2.

State space approach to single molecule localization in fluorescence microscopy

Single molecule super-resolution microscopy enables imaging at sub-diffraction-limit resolution by producing images of subsets of stochastically photoactivated fluorophores over a sequence of frames. In each frame of the sequence, the fluorophores are accurately localized, and the estimated locations are used to construct a high-resolution image of the cellular structures labeled by the fluorophores. Many methods have been developed for localizing fluorophores from the images. The majority of these methods comprise two separate steps: detection and estimation. In the detection step, fluorophores are identified. In the estimation step, the locations of the identified fluorophores are estimated through an iterative approach. Here, we propose a non-iterative state space-based localization method which combines the detection and estimation steps. We demonstrate that the estimated locations obtained from the proposed method can be used as initial conditions in an estimation routine to potentially obtain improved location estimates. The proposed method models the given image as the frequency response of a multi-order system obtained with a balanced state space realization algorithm based on the singular value decomposition of a Hankel matrix. The locations of the poles of the resulting system determine the peak locations in the frequency domain, and the locations of the most significant peaks correspond to the single molecule locations in the original image. The performance of the method is validated using both simulated and experimental data.

Targeting phosphatidylserine with calcium-dependent protein-drug conjugates for the treatment of cancer

In response to cellular stress, phosphatidylserine is exposed on the outer membrane leaflet of tumor blood vessels and cancer cells, motivating the development of phosphatidylserine-specific therapies. The generation of drug-conjugated phosphatidylserine-targeting agents represents an unexplored therapeutic approach, for which antitumor effects are critically dependent on efficient internalization and lysosomal delivery of the cytotoxic drug. In the current study, we have generated phosphatidylserine-targeting agents by fusing phosphatidylserine-binding domains to a human IgG1-derived Fc fragment. The tumor localization and pharmacokinetics of several phosphatidylserine-specific Fc fusions have been analyzed in mice and demonstrate that Fc-Syt1, a fusion containing the synaptotagmin 1 C2A domain, effectively targets tumor tissue. Conjugation of Fc-Syt1 to the cytotoxic drug monomethyl auristatin E results in a protein–drug conjugate (PDC) that is internalized into target cells and, due to the Ca2+ dependence of phosphatidylserine binding, dissociates from phosphatidylserine in early endosomes. The released PDC is efficiently delivered to lysosomes and has potent antitumor effects in mouse xenograft tumor models. Interestingly, although an engineered, tetravalent Fc-Syt1 fusion shows increased binding to target cells, this higher avidity variant demonstrates reduced persistence and therapeutic effects compared with bivalent Fc-Syt1. Collectively, these studies show that finely tuned, Ca2+-switched phosphatidylserine-targeting agents can be therapeutically efficacious. Mol Cancer Ther; 17(1); 169–82. ©2017 AACR.

Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context

Three-dimensional (3D) single molecule fluorescence microscopy affords the ability to investigate subcellular trafficking at the level of individual molecules. An imaged single molecule trajectory, however, often reveals only limited information about the underlying biological process when insufficient information is available about the organelles and other cellular structures with which the molecule interacts. A new 3D fluorescence microscopy imaging modality is described here that enables the simultaneous imaging of the trajectories of fast-moving molecules and the associated cellular context. The new modality is called remote focusing multifocal plane microscopy (rMUM), as it extends multifocal plane microscopy (MUM) with a remote focusing module. MUM is a modality that uses multiple detectors to image distinct focal planes within the specimen at the same time, and it has been demonstrated to allow the determination of 3D single molecule trajectories with high accuracy. Remote focusing is a method that makes use of two additional objective lenses to enable the acquisition of a z-stack of the specimen without having to move the microscope’s objective lens or sample stage, components which are required by MUM to be fixed in place. rMUM’s remote focusing module thus allows the cellular context to be imaged in the form of z-stacks as the trajectories of molecules or other objects of interest are imaged by MUM. In addition to a description of the modality, a discussion of rMUM data analysis and an example of data acquired using an rMUM setup are provided in this paper.

Design and performance of a THz block camera with a 130nm CMOS focal plane array

Recent advances in 130 nm CMOS based Schottky barrier diode THz power detectors enable relatively simple, highperformance focal plane arrays. We present a low size, weight and power block camera which uses polymer refractive optics and a 6x6 focal plane array to image the return from an active source operating at 218 GHz. The operating frequency is chosen for multiple reasons: to coincide with atmospheric transmission windows, to image through degraded visual environments, and to leverage recently developed high power sources available at the Naval Research Laboratory. The sensor achieves better than 30 pW/√Hz NEP at video frame rates while lock-in detecting a modulated source. The three and a half pound camera houses a COTs aspheric polymer optic, detector array, signal amplification and lock-in detection, and outputs data over an Ethernet connection. We will present the camera design, performance metrics, and sample imagery