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?
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 to thin the cells, 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.
Many of our studies use the unicellular green alga Chlamydomonas reinhardtii, which has intricate and reproducible organelle architecture as well as superb cryo-EM imaging properties. In addition, we explore a variety of cellular environments, including mammalian cells, yeast, several species of marine algae, and the ameoboflagellate Naegleria gruberi. We are especially fond of photosynthetic organisms, as we seek to understand how the light-harvesting and CO2-fixing compartments of the chloroplast are affected by environmental stress and climate change.
Recent and ongoing studies in our group include:
We observed that proteasomes tether to two specific sites at the nuclear pore complex (NPC): the inner nuclear membrane and the NPC basket (Albert et al., PNAS 2017). Current efforts are focused on identifying the tethering proteins and investigating the functions of these tethered proteasomes. In collaboration with the Beck group (EMBL), we also found that the Chlamydomonas NPC has an unprecedented oligomeric state, raising questions about the evolution of this fundamental eukaryotic structure (Mosalaganti et al., Nat Comm 2018).
In Chlamydomonas, we resolved a structure of the ribosome bound to the native ER translocon. Comparison of this structure to the human translocon-bound ribosome revealed differences in the TRAP complex that allowed us to dissect the molecular architecture of human TRAP (Pfeffer et al., Nat Comm 2017). We also discovered that proteasomes and Cdc48 cluster together in microcompartments that directly contact the ER membrane. We propose that these microcompartments form by phase separation and help facilitate efficient ER-associated degradation (Albert et al., PNAS 2020).
Golgi and Endosomes
We identified arrays of linker proteins within the Golgi cisternae that likely maintain Golgi architecture and direct cargo transport (Engel et al., PNAS 2015). In collaboration with the Briggs group (MRC-LMB), we resolved the native structures of the COPI and Retromer membrane coats and found that their molecular architecture is highly conserved between eukaryotes (Bykov et al., eLife 2017; Kovtun et al., Nature 2018).
In an earlier study, we visualized the native architecture of thylakoid membranes and described tubule structures that connect the thylakoids and stroma with the matrix of the pyrenoid, which is densely packed with CO2-fixing Rubisco enzymes (Engel et al., eLife 2015). We are building on this study in a few ways: 1) We are mapping all four major complexes of the photosynthetic light reactions into the native thylakoid architecture. 2) In collaboration with the Jonikas group (Princeton), we analyzed the packing of Rubisco within the pyrenoid and discovered that the pyrenoid behaves like a phase-separated liquid droplet (Freeman Rosenzweig et al., Cell 2017). 3) As pyrenoids have independently evolved multiple times, we are exploring pyrenoid organization in a variety of ecologically important marine algae, including diatoms and dinoflagellates. As members of the DFG research group FOR2092, we collaborate with Jörg Nickelsen (LMU München), Michael Schroda (TU Kaiserslautern), and Marc Nowaczyk (Ruhr-Universität Bochum) on mechanisms of thylakoid biogenesis in both cyanobacteria and green algae. Together, we have published studies spanning from isolated thylakoid proteins (Schuller et al., Science 2019; Theis et al., Scientific Reports 2019) to the molecular architecture of thylakoids within the cell (Rast et al., Nat Plants 2019).
Centrioles and Cilia
Using Naegleria, Chlamydomonas, Paramecium, and mammalian cells, we collaborate with the Guichard/Hamel group (Univ. Geneva) to explore how centrioles assemble in diverse evolutionary lineages (Le Guennec et al., Science Advances 2020). We are also investigating how centrioles template the assembly of cilia and how protein traffic is gated between the cell body and ciliary compartment.
In addition to our studies describing ER-associated proteasome clusters and the liquid-like nature of the pyrenoid compartment, we collaborated with the Holt group (NYU) to reveal that mTORC1 signaling regulates phase separation by tuning cytosolic ribosome concentration (Delarue et al., Cell 2018). The ER-associated proteasome clusters (described above) are also likely formed by phase separation.
Focused ion beam milling of a Chlamydomonas cell
Proteasomes tether to two distinct sites at the NPC
Native architecture of the Chlamydomonas Golgi
Liquid-like organization of Rubisco within the pyrenoid
Cyanobacterial thylakoids form "thylapse" contacts
with the plasma membrane