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

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling sub-nanometer 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 prior to 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 seconds of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level. [Media: see text].

Phosphatase to kinase switch of a critical enzyme contributes to timing of cell differentiation.

IMPORTANCE: The process of cell differentiation is highly regulated in both prokaryotic and eukaryotic organisms. The aquatic bacterium, Caulobacter crescentus, undergoes programmed cell differentiation from a motile swarmer cell to a stationary stalked cell with each cell cycle. This critical event is regulated at multiple levels. Kinase activity of the bifunctional enzyme, PleC, is limited to a brief period when it initiates the molecular signaling cascade that results in cell differentiation. Conversely, PleC phosphatase activity is required for pili formation and flagellar rotation. We show that PleC is localized to the flagellar pole by the scaffold protein, PodJ, which is known to suppress PleC kinase activity in vitro. PleC mutants that are unable to bind PodJ have increased kinase activity in vivo, resulting in premature differentiation. We propose a model in which PodJ regulation of PleC's enzymatic activity contributes to the robust timing of cell differentiation during the Caulobacter cell cycle.

The PHB Granule Biogenesis Pathway in Caulobacter.

PHB granules are bacterial organelles that store excess carbohydrates in the form of water-insoluble polyhydroxybutyrate (PHB). The PHB polymerase, phasin (a small amphipathic protein), and active PHB synthesis are essential for the formation of mature PHB granules in Caulobacter crescentus. Granule formation was found to be initiated by the condensation of self-associating PHB polymerase-GFP into foci, closely followed by the recruitment and condensation of phasin-mCherry. Following the active synthesis of PHB and granule maturation, the polymerase dissociates from mature granules and the PHB depolymerase is recruited to the granule. The polymerase directly binds phasin in vitro through its intrinsically disordered N-terminal domain. Thus, granule biogenesis is initiated and controlled by the action of a PHB polymerase and an associated helper protein, phasin, that together synthesize the hydrophobic granule's content while forming the granules protein boundary.