Studies of the Kinetics of Cell Cycle Processes in S. Cerevisiae: The Molecular Basis of Start Irreversibility and Cyclin-Cdk Ordering of Mitotic Events

The cell cycle machinery of Saccharomyces cerevisiae consists of a central negative feedback oscillator comprising cyclin-CDK and its antagonist, APCCdc20. This oscillator is stabilized and tuned by positive feedback loops, and its frequency is modulated by checkpoint controls. Either by directly triggering events, or by entraining independent oscillators controlling events, the cyclin-CDK oscillator regulates the key events of the cell cycle. These events have an established order and timing within the overall cycle. The work I describe in this thesis concerns two fundamental questions: how is the order and timing of cell cycle events controlled, and what sets the intrinsic frequency of the cell cycle oscillator? I describe work on two major processes in the cell division cycle that reveals two very different modes of regulation. The first of these processes – Start – represents a pivotal commitment to divide. In collaboration with Gilles Charvin, I demonstrate that positive feedback in the molecular machinery underlying Start acts as a bistable switch that renders this regulatory transition irreversible. The second major process is Mitosis, a set of events all triggered by the same class of cyclin-CDKs and yet occurring in a set and reproducible order. I describe an ordering mechanism underlying this choreography that relies on the natural ramping-up of cyclin-CDK activity level. The observation that different events require different levels of cyclin-CDK activity leads to the question of how these thresholds are set. To begin to answer this, I discuss how mitotic cyclin-CDK triggers two different events – depolarization of growth and formation of the mitotic spindle – in two very different ways. The first relies on entrainment of an independent oscillator controlling growth polarization; the other may involve the simultaneous regulation of multiple targets. The observation that cyclin-CDK is rate-limiting for mitotic events suggests that increasing the level of this key cell cycle regulator above its endogenous range should accelerate Mitosis, and I show evidence that this is the case. Quite surprisingly, this increase in cyclin-CDK abundance also accelerates the frequency of the cell cycle oscillator as a whole through its effect on growth. This provides an intriguing new answer to the question of what sets the intrinsic frequency of the cell cycle oscillator. Together, this work underscores the central role of the mitotic cyclin-CDK regulator, which controls not only the relative timing of individual cell cycle events, but also the growth rate of the cell, and the overall frequency of the cell cycle oscillator

A new view into prokaryotic cell biology from electron cryotomography

Electron cryotomography (ECT) enables intact cells to be visualized in 3D in an essentially native state to 'macromolecular' (~4 nm) resolution, revealing the basic architectures of complete nanomachines and their arrangements in situ. Since its inception, ECT has advanced our understanding of many aspects of prokaryotic cell biology, from morphogenesis to subcellular compartmentalization and from metabolism to complex interspecies interactions. In this Review, we highlight how ECT has provided structural and mechanistic insights into the physiology of bacteria and archaea and discuss prospects for the future

The Caltech Tomography Database and Automatic Processing Pipeline

Here we describe the Caltech Tomography Database and automatic image processing pipeline, designed to process, store, display, and distribute electron tomographic data including tilt-series, sample information, data collection parameters, 3D reconstructions, correlated light microscope images, snapshots, segmentations, movies, and other associated files. Tilt-series are typically uploaded automatically during collection to a user’s “Inbox” and processed automatically, but can also be entered and processed in batches via scripts or file-by-file through an internet interface. As with the video website YouTube, each tilt-series is represented on the browsing page with a link to the full record, a thumbnail image and a video icon that delivers a movie of the tomogram in a pop-out window. Annotation tools allow users to add notes and snapshots. The database is fully searchable, and sets of tilt-series can be selected and re-processed, edited, or downloaded to a personal workstation. The results of further processing and snapshots of key results can be recorded in the database, automatically linked to the appropriate tilt-series. While the database is password-protected for local browsing and searching, datasets can be made public and individual files can be shared with collaborators over the Internet. Together these tools facilitate high-throughput tomography work by both individuals and groups

Electron Cryotomography of Bacterial Secretion Systems

In biology, function arises from form. For bacterial secretion systems, which often span two membranes, avidly bind to the cell wall, and contain hundreds of individual proteins, studying form is a daunting task, made possible by electron cryotomography (ECT). ECT is the highest-resolution imaging technique currently available to visualize unique objects inside cells, providing a three-dimensional view of the shapes and locations of large macromolecular complexes in their native environment. Over the past 15 years, ECT has contributed to the study of bacterial secretion systems in two main ways: by revealing intact forms for the first time and by mapping components into these forms. Here we highlight some of these contributions, revealing structural convergence in type II secretion systems, structural divergence in type III secretion systems, unexpected structures in type IV secretion systems, and unexpected mechanisms in types V and VI secretion systems. Together, they offer a glimpse into a world of fantastic forms—nanoscale rotors, needles, pumps, and dart guns—much of which remains to be explored

Plasma membrane damage removal by F-actin-mediated shedding from repurposed filopodia

Repairing plasma membrane damage is vital to eukaryotic cell survival. Membrane shedding is thought to be key to this repair process, but a detailed view of how the process occurs is still missing. Here we used electron cryotomography to image the ultrastructural details of plasma membrane wound healing. We found that filopodia-like protrusions are built at damage sites, accompanied by retraction of neighboring filopodia, and that these repurposed protrusions act as scaffolds for membrane shedding. This suggests a new role for filopodia as reservoirs of membrane and actin for plasma membrane damage repair. Damage-induced shedding was dependent on F-actin dynamics and Myo1a, as well as Vps4B, an important component of the ESCRT machinery. Thus we find that damage shedding is more complex than current models of simple vesiculation from flat membrane domains. Rather, we observe structural similarities between damage-mediated shedding and constitutive shedding from enterocytes that argue for conservation of a general membrane shedding mechanism

ETDB-Caltech: a blockchain-based distributed public database for electron tomography

Three-dimensional electron microscopy techniques like electron tomography provide valuable insights into cellular structures, and present significant challenges for data storage and dissemination. Here we explored a novel method to publicly release more than 11,000 such datasets, more than 30 TB in total, collected by our group. Our method, based on a peer-to-peer file sharing network built around a blockchain ledger, offers a distributed solution to data storage. In addition, we offer a user-friendly browser-based interface, https://etdb.caltech.edu, for anyone interested to explore and download our data. We discuss the relative advantages and disadvantages of this system and provide tools for other groups to mine our data and/or use the same approach to share their own imaging datasets

Structural conservation of chemotaxis machinery across Archaea and Bacteria

Chemotaxis allows cells to sense and respond to their environment. In Bacteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two-component regulatory system. These arrays take the form of highly stereotyped super-lattices comprising hexagonally packed trimers-of-receptor-dimers networked by rings of histidine kinase and coupling proteins. This structure is conserved across chemotactic Bacteria, and between membrane-bound and cytoplasmic arrays, and gives rise to the highly cooperative, dynamic nature of the signalling system. The chemotaxis system, absent in eukaryotes, is also found in Archaea, where its structural details remain uncharacterized. Here we provide evidence that the chemotaxis machinery was not present in the last archaeal common ancestor, but rather was introduced in one of the waves of lateral gene transfer that occurred after the branching of Eukaryota but before the diversification of Euryarchaeota. Unlike in Bacteria, the chemotaxis system then evolved largely vertically in Archaea, with very few subsequent successful lateral gene transfer events. By electron cryotomography, we find that the structure of both membrane-bound and cytoplasmic chemoreceptor arrays is conserved between Bacteria and Archaea, suggesting the fundamental importance of this signalling architecture across diverse prokaryotic lifestyles

Simulations suggest a constrictive force is required for Gram-negative bacterial cell division

To divide, Gram-negative bacterial cells must remodel cell wall at the division site. It remains debated, however, whether this cell wall remodeling alone can drive membrane constriction, or if a constrictive force from the tubulin homolog FtsZ is required. Previously, we constructed software (REMODELER 1) to simulate cell wall remodeling during growth. Here, we expanded this software to explore cell wall division (REMODELER 2). We found that simply organizing cell wall synthesis complexes at the midcell is not sufficient to cause invagination, even with the implementation of a make-before-break mechanism, in which new hoops of cell wall are made inside the existing hoops before bonds are cleaved. Division can occur, however, when a constrictive force brings the midcell into a compressed state before new hoops of relaxed cell wall are incorporated between existing hoops. Adding a make-before-break mechanism drives division with a smaller constrictive force sufficient to bring the midcell into a relaxed, but not necessarily compressed, state