The Interplay Between Organelle Form and Function
Cells accomplish the biochemical reactions of life by concentrating their proteins into a variety of subcellular compartments called organelles. Our group explores the relationship between the form of the organelle and the function of its resident macromolecules. How does organelle architecture direct molecular function, and reciprocally, how do macromolecules sculpt and shape organelles?
Our Approach: Cellular Structural Biology with Cryo-electron Tomography
To investigate these questions, our group uses cryo-electron tomography (cryo-ET) to directly visualize macromolecules “in situ”, within the native cellular environment. First, we rapidly freeze the cells in non-crystalline vitreous ice, preserving them in a state of suspended animation. Next, we use a focused ion beam (FIB) to thin the cells (Schaffer et al. 2017), followed by cryo-ET to acquire 3D images (called tomograms) of the native cellular interior with molecular resolution. These tomograms enable us to solve molecular structures directly within the cell, at sufficient resolution to distinguish different conformational states and interaction partners. We then map these structures back into the cellular volume with nanometer precision, allowing us to analyze molecular organization within the cell at the scale of single molecules.
Cryo-ET is uniquely positioned at the intersection between the scales of Structural Biology and Cell Biology. In our research approach, we seek to integrate multi-modal data across length- and time-scales, leveraging the strengths of different techniques to draw a more comprehensive picture of the systems we study (McCafferty et al. 2024). This includes using cryo-EM for a high-resolution look at isolated complexes, mass spectrometry to generate molecular inventories and reveal interactions, as well as fluorescence microscopy, spectroscopy, and cellular assays to gain insights in to dynamics, cellular-scale organization, and physiological state.
To investigate these questions, our group uses cryo-electron tomography (cryo-ET) to directly visualize macromolecules “in situ”, within the native cellular environment. First, we rapidly freeze the cells in non-crystalline vitreous ice, preserving them in a state of suspended animation. Next, we use a focused ion beam (FIB) to thin the cells (Schaffer et al. 2017), followed by cryo-ET to acquire 3D images (called tomograms) of the native cellular interior with molecular resolution. These tomograms enable us to solve molecular structures directly within the cell, at sufficient resolution to distinguish different conformational states and interaction partners. We then map these structures back into the cellular volume with nanometer precision, allowing us to analyze molecular organization within the cell at the scale of single molecules.
Cryo-ET is uniquely positioned at the intersection between the scales of Structural Biology and Cell Biology. In our research approach, we seek to integrate multi-modal data across length- and time-scales, leveraging the strengths of different techniques to draw a more comprehensive picture of the systems we study (McCafferty et al. 2024). This includes using cryo-EM for a high-resolution look at isolated complexes, mass spectrometry to generate molecular inventories and reveal interactions, as well as fluorescence microscopy, spectroscopy, and cellular assays to gain insights in to dynamics, cellular-scale organization, and physiological state.
Our Questions: Molecular Architecture of Bioenergetic Organelles
We study how the structures of cellular organelles and their embedded molecular complexes dive the conversion of environmental energy into biological energy. We seek a mechanistic understanding of how bioenergetic organelles are built and how they adapt to environmental stresses, including those intensified by climate change. In addition, we are interested in the evolutionary relationships between these organelles and complexes in different species (Perez-Boerema et al. 2024), which can provide insights into origins and fundamental organizing principles.
Thylakoid membranes: During the light reactions of photosynthesis, thylakoid membranes inside chloroplasts and cyanobacteria use the energy of sunlight to split water, liberating protons and electrons that are used to regenerate molecules that carry biological energy and reducing potential: ATP and NADPH. We explore the molecular organization of thylakoids across lineages, including cyanobacteria (Rast et al. 2019), green algae (Wietrzynski et al. 2020), plants (Wietrzynski et al. 2025), and diverse marine algae including diatoms and dinoflagellates. We investigate how thylakoids establish their intricate architecture, which sorts different photosynthetic complexes into distinct membrane domains (e.g., the stacked and non-stacked membranes of plants and many algae). We explore how this molecular organization is remodeled in response to changes in light intensity and color, as well as how it is repaired from photodamage. Furthermore, we study the roles of protein assembly machinery (Waltz et al. 2026) and membrane-remodeling proteins (Gupta et al. 2021) in thylakoid biogenesis and maintenance.
Pyrenoids: During the dark reactions of photosynthesis, ATP and NADPH from the light reactions are used to fix CO2 into sugar, which stores energy until the plant needs it (or we eat it). The enzyme that catalyzes carbon fixation, Rubisco, has slow kinetics and poor selectivity between CO2 and O2. Oxygen poisons the enzyme and wastes energy, in a process known as photorespiration. To improve Rubisco efficiency, algae compartmentalize Rubisco in a chloroplast sub-compartment called the pyrenoid, which concentrates the enzyme together with a high local concentration of its CO2 substrate. Approximately one-third of global carbon fixation is performed by pyrenoids, which are relatively recent innovation in the evolutionary history of photosynthesis and have convergently evolved several times in different lineages. We explore the molecular composition, architecture, and organizing principles of pyrenoids across the diverse algae, including the phase separation of Rubisco in green algae (Freeman Rosenzweig et al. 2017; He et al. 2020; Kumar et al. 2026) and the encapsulation of the diatom pyrenoid in a protein shell (Shimakawa et al. 2024; Nam et al. 2024).
Mitochondria: Both autotrophic and heterotrophic eukaryotes have mitochondria. Environmental energy stored by photosynthesis as sugar can then be broken down into the electron donors NADH and FADH2, which are used in the crista membranes of mitochondria to power proton pumping by respiratory complexes and ultimately the regeneration of ATP by ATP synthase. We explore the eclectic structures of mitochondrial complexes in diverse organisms (Waltz et al. 2021), with a focus on how and why respiratory complexes assemble together into large "respirasomes" (Waltz et al. 2025). We seek to understand how the molecular organization of crista membranes underlies the architecture of these biogenic compartments, which can vary from flat sheets to cylindrical tubes.
Extremophile Prokaryotes: Some anaerobic bacteria and archaea use ancient metabolisms, which are fueled by electrons from environmental sources including molecular hydrogen (H2) and sulfide (H2S). We are interested in exploring the cellular organization of exotic bioenergetic machinery that enables life in extreme environments. One example is our work on enzyme-decorated nanowires that use electrons from H2 to reduce CO2, enabling carbon fixation "at the thermodynamic limit of life" (Dietrich et al 2022).
Cilia: Not a bioenergetic organelle, but a longstanding topic in the lab. We are curious about the structures and assembly mechanisms of these multifunctional eukaryotic compartments, which coordinate motility and signaling across the tree of life. In diverse organisms, we have studied the the architecture of centrioles (Le Guennec et al. 2020, Klena et al. 2020, Bertiaux et al. 2025) as well as the transition zone, which gates entry of proteins at the ciliary base (van den Hoek et al. 2022).
Environmental Structural Biology: We are developing workflows to perform mechanistic cryo-ET studies of organisms sampled directly from the environment. This enable us to explore a wider range of biodiversity (many organisms cannot be cultured) and capture inter-species interactions as well as the influence of changing environmental conditions on organelle architecture and function. To bring advanced methods to the field, we teamed with the Traversing European Coastlines (TREC) mission (Leisch et al. 2026).
We study how the structures of cellular organelles and their embedded molecular complexes dive the conversion of environmental energy into biological energy. We seek a mechanistic understanding of how bioenergetic organelles are built and how they adapt to environmental stresses, including those intensified by climate change. In addition, we are interested in the evolutionary relationships between these organelles and complexes in different species (Perez-Boerema et al. 2024), which can provide insights into origins and fundamental organizing principles.
Thylakoid membranes: During the light reactions of photosynthesis, thylakoid membranes inside chloroplasts and cyanobacteria use the energy of sunlight to split water, liberating protons and electrons that are used to regenerate molecules that carry biological energy and reducing potential: ATP and NADPH. We explore the molecular organization of thylakoids across lineages, including cyanobacteria (Rast et al. 2019), green algae (Wietrzynski et al. 2020), plants (Wietrzynski et al. 2025), and diverse marine algae including diatoms and dinoflagellates. We investigate how thylakoids establish their intricate architecture, which sorts different photosynthetic complexes into distinct membrane domains (e.g., the stacked and non-stacked membranes of plants and many algae). We explore how this molecular organization is remodeled in response to changes in light intensity and color, as well as how it is repaired from photodamage. Furthermore, we study the roles of protein assembly machinery (Waltz et al. 2026) and membrane-remodeling proteins (Gupta et al. 2021) in thylakoid biogenesis and maintenance.
Pyrenoids: During the dark reactions of photosynthesis, ATP and NADPH from the light reactions are used to fix CO2 into sugar, which stores energy until the plant needs it (or we eat it). The enzyme that catalyzes carbon fixation, Rubisco, has slow kinetics and poor selectivity between CO2 and O2. Oxygen poisons the enzyme and wastes energy, in a process known as photorespiration. To improve Rubisco efficiency, algae compartmentalize Rubisco in a chloroplast sub-compartment called the pyrenoid, which concentrates the enzyme together with a high local concentration of its CO2 substrate. Approximately one-third of global carbon fixation is performed by pyrenoids, which are relatively recent innovation in the evolutionary history of photosynthesis and have convergently evolved several times in different lineages. We explore the molecular composition, architecture, and organizing principles of pyrenoids across the diverse algae, including the phase separation of Rubisco in green algae (Freeman Rosenzweig et al. 2017; He et al. 2020; Kumar et al. 2026) and the encapsulation of the diatom pyrenoid in a protein shell (Shimakawa et al. 2024; Nam et al. 2024).
Mitochondria: Both autotrophic and heterotrophic eukaryotes have mitochondria. Environmental energy stored by photosynthesis as sugar can then be broken down into the electron donors NADH and FADH2, which are used in the crista membranes of mitochondria to power proton pumping by respiratory complexes and ultimately the regeneration of ATP by ATP synthase. We explore the eclectic structures of mitochondrial complexes in diverse organisms (Waltz et al. 2021), with a focus on how and why respiratory complexes assemble together into large "respirasomes" (Waltz et al. 2025). We seek to understand how the molecular organization of crista membranes underlies the architecture of these biogenic compartments, which can vary from flat sheets to cylindrical tubes.
Extremophile Prokaryotes: Some anaerobic bacteria and archaea use ancient metabolisms, which are fueled by electrons from environmental sources including molecular hydrogen (H2) and sulfide (H2S). We are interested in exploring the cellular organization of exotic bioenergetic machinery that enables life in extreme environments. One example is our work on enzyme-decorated nanowires that use electrons from H2 to reduce CO2, enabling carbon fixation "at the thermodynamic limit of life" (Dietrich et al 2022).
Cilia: Not a bioenergetic organelle, but a longstanding topic in the lab. We are curious about the structures and assembly mechanisms of these multifunctional eukaryotic compartments, which coordinate motility and signaling across the tree of life. In diverse organisms, we have studied the the architecture of centrioles (Le Guennec et al. 2020, Klena et al. 2020, Bertiaux et al. 2025) as well as the transition zone, which gates entry of proteins at the ciliary base (van den Hoek et al. 2022).
Environmental Structural Biology: We are developing workflows to perform mechanistic cryo-ET studies of organisms sampled directly from the environment. This enable us to explore a wider range of biodiversity (many organisms cannot be cultured) and capture inter-species interactions as well as the influence of changing environmental conditions on organelle architecture and function. To bring advanced methods to the field, we teamed with the Traversing European Coastlines (TREC) mission (Leisch et al. 2026).
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Our Methods Development: Towards Visual Proteomics
Cryo-ET data is incredibly rich in information. The goal of visual proteomics is to resolve or predict the structure, functional state, and interactions of every molecular complex visualized inside native cells. This approach promises to be transformative for biology, but has many challenges posed by the diverse molecular composition of cells and noisy, undersampled cryo-ET data. To enable a richer cryo-ET analysis, we are developing computational methods in tomogram reconstruction and denoising (cryoLithe: Kishore et al. 2025; ICECREAM: Kishore et al. 2025), as well as membrane segmentation and particle picking (MemBrain: Lamm et al. 2022, Lamm et al. 2024; Surforama: Yamauchi et al. 2024; DeepFinder: Moebel et al. 2021). We also helped develop a tool to predict protein complex stoichiometry (Stoic: Litvinov et al. 2026), with the goal of improving structure discovery inside cells. We are committed to open sharing of cryo-ET data and annotations to empower both biological discovery and method development. One leading example is the Chlamydomonas community dataset (Kelley et al. 2026). See our RESOURCES page for more on software, tutorials, and publicly shared data. |
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