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Time-resolved cryogenic electron tomography for the study of transient cellular processes

Yoniles, Joseph ; Summers, Jacob A. ; Zielinski, Kara A. ; Antolini, Cali ; Panjalingam, Mayura ; Lisova, Stella ; Moss, Frank R. ; Di Perna, Maximus Aldo ; Kupitz, Christopher ; Hunter, Mark S. ; Pollack, Lois ; Wakatsuki, Soichi ; Dahlberg, Peter D. Garner, Ethan

Molecular biology of the cell, 2024-07, Vol.35 (7), p.mr4-mr4 [Periódico revisado por pares]

United States: American Society for Cell Biology

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  • Título:
    Time-resolved cryogenic electron tomography for the study of transient cellular processes
  • Autor: Yoniles, Joseph ; Summers, Jacob A. ; Zielinski, Kara A. ; Antolini, Cali ; Panjalingam, Mayura ; Lisova, Stella ; Moss, Frank R. ; Di Perna, Maximus Aldo ; Kupitz, Christopher ; Hunter, Mark S. ; Pollack, Lois ; Wakatsuki, Soichi ; Dahlberg, Peter D.
  • Garner, Ethan
  • Assuntos: BASIC BIOLOGICAL SCIENCES
  • É parte de: Molecular biology of the cell, 2024-07, Vol.35 (7), p.mr4-mr4
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
    National Science Foundation (NSF)
    USDOE Laboratory Directed Research and Development (LDRD) Program
    AC02-76SF00515; DGE-1656518; S10-OD021600; R35GM118067; T32 GM136568; P41GM139687; 1231306
    USDOE Office of Science (SC), Basic Energy Sciences (BES)
    National Institutes of Health (NIH)
  • Descrição: Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling subnanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second before freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 s of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a subsecond temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level. Capturing cellular dynamics with millisecond precision and nanometer-scale spatial resolution is an open challenge and of critical importance due to the many cellular processes that occur on these time and length scales. The sample preparation method presented here enables the rapid mixing of cellular samples with solution-phase stimulants followed by rapid freezing at well-controlled time points. Numerous methods of interrogation can be performed following freezing, including cryogenic electron tomography, which enables the visualization of dynamic cellular processes with unprecedented simultaneous spatial and temporal resolution.
  • Editor: United States: American Society for Cell Biology
  • Idioma: Inglês
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