Kevin Rose

All Publications

• In situ cryo- ET visualization of mitochondrial depolarization and mitophagic engulfment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rose, K., Herrmann, E., Kakudji, E., Lizarrondo, J., Celebi, A., Wilfling, F., Lewis, S. C., Hurley, J. H. 2025; 122 (31): e2511890122

  Abstract

  Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson's disease by mutations in PINK1 and PRKN. Parkin-expressing U2 osteosarcoma (U2OS) cells were treated with the depolarizing agents oligomycin and antimycin A (OA) and subjected to cryo-focused ion beam milling and in situ cryo-electron tomography. Mitochondria were fragmented and devoid of matrix calcium phosphate crystals. Phagophores were visualized, with bridge-like lipid transporter densities connected to mitophagic phagophores. A subpopulation of ATP synthases relocalized from cristae to the inner boundary membrane. The structure of the dome-shaped prohibitin complex, a dodecamer of PHB1-PHB2 dimers, was determined in situ by subtomogram averaging in untreated and treated cells and found to exist in open and closed conformations, with the closed conformation being enriched by OA treatment. These findings provide a set of native snapshots of the manifold nano-structural consequences of mitochondrial depolarization and provide a baseline for future in situ dissection of Parkin-dependent mitophagy.

  View details for DOI 10.1073/pnas.2511890122

  View details for Web of Science ID 001547036900001

  View details for PubMedID 40743392

  View details for PubMedCentralID PMC12337332

• A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy. The Journal of cell biology Tudorica, D. A., Basak, B., Puerta Cordova, A. S., Khuu, G., Rose, K., Lazarou, M., Holzbaur, E. L., Hurley, J. H. 2024; 223 (7)

  Abstract

  Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

  View details for DOI 10.1083/jcb.202309015

  View details for PubMedID 38728007

  View details for PubMedCentralID PMC11090050

• Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes. Proceedings of the National Academy of Sciences of the United States of America Rose, K., Jepson, T., Shukla, S., Maya-Romero, A., Kampmann, M., Xu, K., Hurley, J. H. 2024; 121 (22): e2315690121

  Abstract

  The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

  View details for DOI 10.1073/pnas.2315690121

  View details for PubMedID 38781206

  View details for PubMedCentralID PMC11145263

• In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair. Proceedings of the National Academy of Sciences of the United States of America Shukla, S., Larsen, K. P., Ou, C., Rose, K., Hurley, J. H. 2022; 119 (35): e2205590119

  Abstract

  The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.

  View details for DOI 10.1073/pnas.2205590119

  View details for PubMedID 35994655

  View details for PubMedCentralID PMC9436306

• When in Need of an ESCRT: The Nature of Virus Assembly Sites Suggests Mechanistic Parallels between Nuclear Virus Egress and Retroviral Budding (vol 13, 1138, 2021) VIRUSES-BASEL Rose, K. M., Spada, S. J., Hirsch, V. M., Bouamr, F. 2021; 13 (9)

  Abstract

  The authors wish to make the following erratum to this paper [...].

  View details for DOI 10.3390/v13091705

  View details for Web of Science ID 000701272500001

  View details for PubMedID 34578469

  View details for PubMedCentralID PMC8472907

• From Capsids to Complexes: Expanding the Role of TRIM5α in the Restriction of Divergent RNA Viruses and Elements. Viruses Rose, K. M., Spada, S. J., Broeckel, R., McNally, K. L., Hirsch, V. M., Best, S. M., Bouamr, F. 2021; 13 (3)

  Abstract

  An evolutionary arms race has been ongoing between retroviruses and their primate hosts for millions of years. Within the last century, a zoonotic transmission introduced the Human Immunodeficiency Virus (HIV-1), a retrovirus, to the human population that has claimed the lives of millions of individuals and is still infecting over a million people every year. To counteract retroviruses such as this, primates including humans have evolved an innate immune sensor for the retroviral capsid lattice known as TRIM5α. Although the molecular basis for its ability to restrict retroviruses is debated, it is currently accepted that TRIM5α forms higher-order assemblies around the incoming retroviral capsid that are not only disruptive for the virus lifecycle, but also trigger the activation of an antiviral state. More recently, it was discovered that TRIM5α restriction is broader than previously thought because it restricts not only the human retroelement LINE-1, but also the tick-borne flaviviruses, an emergent group of RNA viruses that have vastly different strategies for replication compared to retroviruses. This review focuses on the underlying mechanisms of TRIM5α-mediated restriction of retroelements and flaviviruses and how they differ from the more widely known ability of TRIM5α to restrict retroviruses.

  View details for DOI 10.3390/v13030446

  View details for PubMedID 33801908

  View details for PubMedCentralID PMC7998678

• Budding of a Retrovirus: Some Assemblies Required. Viruses Rose, K. M., Hirsch, V. M., Bouamr, F. 2020; 12 (10)

  Abstract

  One of the most important steps in any viral lifecycle is the production of progeny virions. For retroviruses as well as other viruses, this step is a highly organized process that occurs with exquisite spatial and temporal specificity on the cellular plasma membrane. To facilitate this process, retroviruses encode short peptide motifs, or L domains, that hijack host factors to ensure completion of this critical step. One such cellular machinery targeted by viruses is known as the Endosomal Sorting Complex Required for Transport (ESCRTs). Typically responsible for vesicular trafficking within the cell, ESCRTs are co-opted by the retroviral Gag polyprotein to assist in viral particle assembly and release of infectious virions. This review in the Viruses Special Issue "The 11th International Retroviral Nucleocapsid and Assembly Symposium", details recent findings that shed light on the molecular details of how ESCRTs and the ESCRT adaptor protein ALIX, facilitate retroviral dissemination at sites of viral assembly.

  View details for DOI 10.3390/v12101188

  View details for PubMedID 33092109

  View details for PubMedCentralID PMC7589157

• A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission. Nature structural & molecular biology Flower, T. G., Takahashi, Y., Hudait, A., Rose, K., Tjahjono, N., Pak, A. J., Yokom, A. L., Liang, X., Wang, H. G., Bouamr, F., Voth, G. A., Hurley, J. H. 2020; 27 (6): 570-580

  Abstract

  The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, multivesicular body biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101-VPS28-VPS37B-MVB12A was determined, revealing an ESCRT-I helical assembly with a 12-molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse-grained (CG) simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor; it has an essential scaffolding and mechanical role in its own right.

  View details for DOI 10.1038/s41594-020-0426-4

  View details for PubMedID 32424346

  View details for PubMedCentralID PMC7339825

• Selection of 2'-Deoxy-2'-Fluoroarabino Nucleic Acid (FANA) Aptamers That Bind HIV-1 Integrase with Picomolar Affinity. ACS chemical biology Rose, K. M., Alves Ferreira-Bravo, I., Li, M., Craigie, R., Ditzler, M. A., Holliger, P., DeStefano, J. J. 2019; 14 (10): 2166-2175

  Abstract

  Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2'-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of ∼50-100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5' DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2'-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.

  View details for DOI 10.1021/acschembio.9b00237

  View details for PubMedID 31560515

  View details for PubMedCentralID PMC7005942