Dara Dowlatshahi

All Publications

• Magnetic resonance control of reaction yields through genetically-encoded protein:flavin spin-correlated radicals in a live animal. bioRxiv : the preprint server for biology Burd, S. C., Bagheri, N., Ingaramo, M., Condon, A. F., Mondal, S., Dowlatshahi, D. P., Summers, J. A., Mukherjee, S., York, A. G., Wakatsuki, S., Boxer, S. G., Kasevich, M. 2025

  Abstract

  Radio-frequency (RF) magnetic fields can influence reactions involving spin-correlated radical pairs. This provides a mechanism by which RF fields can influence living systems at the biomolecular level. Here we report the modification of the emission of various red fluorescent proteins (RFPs), in the presence of a flavin cofactor, induced by a combination of static and RF magnetic fields. Resonance features in the protein fluorescence intensity were observed near the electron spin resonance frequency at the corresponding static magnetic field strength. This effect was measured at room temperature both in vitro and in the nematode C. elegans , genetically modified to express the RFP mScarlet. These observations suggest that the magnetic field effects measured in RFP-flavin systems are due to quantum-correlated radical pairs. Our experiments demonstrate that RF magnetic fields can influence dynamics of reactions involving RFPs in biologically relevant conditions, and even within a living animal. These results have implications for the development of a new class of genetic tools based on RF manipulation of genetically-encoded quantum systems.

  View details for DOI 10.1101/2025.02.27.640669

  View details for PubMedID 40093161

  View details for PubMedCentralID PMC11908193

• Engineered Immunomodulatory Extracellular Vesicles from Epithelial Cells with the Capacity for Stimulation of Innate and Adaptive Immunity in Cancer and Autoimmunity ACS NANO Luo, X., Kugeratski, F. G., Dowlatshahi, D. P., Sugimoto, H., Arian, K. A., Fan, Y., Huang, L., Wills, D., Lilla, S., Hodge, K., Zanivan, S. R., Lebleu, V. S., Mcandrews, K. M., Kalluri, R. 2025; 19 (5): 5193-5216

  Abstract

  Extracellular vesicles (EVs) are generated in all cells. Systemic administration of allogenic EVs derived from epithelial and mesenchymal cells has been shown to be safe, despite carrying an array of functional molecules, including thousands of proteins. To address whether epithelial cell-derived EVs can be modified to acquire the capacity to induce an immune response, we engineered 293T EVs to harbor the immunomodulatory molecules CD80, OX40L, and PD-L1. We demonstrated abundant levels of these proteins in the engineered cells and EVs. Functionally, the engineered EVs efficiently elicited positive and negative costimulation of human and murine T cells. In the setting of cancer and autoimmune hepatitis, the engineered EVs modulated T cell functions and altered disease progression. OX40L EVs also provided enhanced antitumor activity in combination with anti-CTLA-4 in melanoma-bearing mice. In addition, we added multiple immunomodulatory proteins in EVs (EVmIM), attempting to elicit an immune response in both lymphoid and myeloid compartments. The EVmIM containing CD80, 4-1BBL, CD40L, CD2, and CD32 engaged both T cells and antigen presenting cells (APCs) in melanoma tumors, demonstrating the capacity for EVmIM to elicit antitumor activity. Our work provides evidence that EVs can be engineered to induce specific immune responses with translational potential to modulate immune cell functions in pathological settings.

  View details for DOI 10.1021/acsnano.4c09688

  View details for Web of Science ID 001406985200001

  View details for PubMedID 39869047

  View details for PubMedCentralID PMC12043189

• Exploring Transient States of PAmKate to Enable Improved Cryogenic Single-Molecule Imaging. Journal of the American Chemical Society Perez, D., Dowlatshahi, D. P., Azaldegui, C. A., Ansell, T. B., Dahlberg, P. D., Moerner, W. E. 2024

  Abstract

  Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of 2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.

  View details for DOI 10.1021/jacs.4c05632

  View details for PubMedID 39388715

• Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging. bioRxiv : the preprint server for biology Perez, D., Dowlatshahi, D. P., Azaldegui, C. A., Dahlberg, P. D., Moerner, W. E. 2024

  Abstract

  Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nano-scale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) which photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remains a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find a transient state forms with a lifetime of ~2 ms which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for study of other FPs at cryogenic temperatures.

  View details for DOI 10.1101/2024.04.24.590965

  View details for PubMedID 38712218

  View details for PubMedCentralID PMC11071506

• ALIX Is a Lys63-Specific Polyubiquitin Binding Protein that Functions in Retrovirus Budding DEVELOPMENTAL CELL Dowlatshahi, D. P., Sandrin, V., Vivona, S., Shaler, T. A., Kaiser, S. E., Melandri, F., Sundquist, W. I., Kopito, R. R. 2012; 23 (6): 1247-1254

  Abstract

  The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release.

  View details for DOI 10.1016/j.devcel.2012.10.023

  View details for Web of Science ID 000312429200020

  View details for PubMedID 23201121

  View details for PubMedCentralID PMC3522770

• An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C-elegans CURRENT BIOLOGY Greer, E. L., Dowlatshahi, D., Banko, M. R., Villen, J., Hoang, K., Blanchard, D., Gygi, S. P., Brunet, A. 2007; 17 (19): 1646-1656

  Abstract

  Dietary restriction (DR) is the most effective environmental intervention to extend lifespan in a wide range of species. However, the molecular mechanisms underlying the benefits of DR on longevity are still poorly characterized. AMP-activated protein kinase (AMPK) is activated by a decrease in energy levels, raising the possibility that AMPK might mediate lifespan extension by DR.By using a novel DR assay that we developed and validated in C. elegans, we find that AMPK is required for this DR method to extend lifespan and delay age-dependent decline. We find that AMPK exerts its effects in part via the FOXO transcription factor DAF-16. FOXO/DAF-16 is necessary for the beneficial effects of this DR method on lifespan. Expression of an active version of AMPK in worms increases stress resistance and extends longevity in a FOXO/DAF-16-dependent manner. Lastly, we find that AMPK activates FOXO/DAF-16-dependent transcription and phosphorylates FOXO/DAF-16 at previously unidentified sites, suggesting a possible direct mechanism of regulation of FOXO/DAF-16 by AMPK.Our study shows that an energy-sensing AMPK-FOXO pathway mediates the lifespan extension induced by a novel method of dietary restriction in C. elegans.

  View details for DOI 10.1016/j.cub.2007.08.047

  View details for Web of Science ID 000250125200023

  View details for PubMedID 17900900

  View details for PubMedCentralID PMC2185793