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:

Nucleus
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 (MPI Biophysics), we also found that the Chlamydomonas NPC has a dilated central channel and an unprecedented oligomeric state, raising questions about the evolution of this fundamental eukaryotic structure (Mosalaganti et al., Nat Comm 2018).

Endoplasmic Reticulum
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 Sorting Organelles
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 (MPI Biochemistry), 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).

Chloroplast
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 continue to build on this work, and now the chloroplast is a central topic of study in our lab.

1) Thylakoid membranes: In Chlamydomonas, we mapped all four major complexes of the photosynthetic light reactions into the native thylakoid architecture, revealing strict lateral heterogeneity between stacked and unstacked regions (Wietrzynski et al., eLife 2020).  We now aim to apply these tools to understand how thylakoid architecture in plants and algae is remodeled in response to changing environmental conditions.  We collaborated with the Nickelsen group (LMU Munich) to visualize the native architecture of thylakoids in the cyanobacterium Synechocystis, revealing contact sites between the thylakoids and plasma membrane that may be involved in thylakoid biogenesis (Rast et al., Nat Plants 2019).  We teamed up with the Schuller group (SYNMICRO Marburg) and Nowaczyk group (Univ. Bochum) on the high-resolution structures of photosynthetic membrane complexes (Schuller et al., Science 2019; Zabret et al., Nat Plants 2021).  In collaboration with the Schroda group (TU Kaiserslautern), we have performed detailed studies of the membrane remodeling protein VIPP1, the photosynthetic homolog of ESCRT-III, which is required for the biogenesis and maintenance of thylakoid membranes.  We solved high-resolution VIPP1 structures, revealed the mechanisms of nucleotide hydrolysis and lipid binding, and demonstrated how this specific lipid interaction is required in vivo to maintain the structural integrity of thylakoids under high-light stress (Theis et al., Sci Rep 2019; Gupta et al, Cell 2021).

2) Pyrenoids:  In collaboration with the Jonikas group (Princeton), we analyzed the packing of Rubisco within the Chlamydomonas pyrenoid and discovered that the pyrenoid behaves like a phase-separated liquid droplet (Freeman Rosenzweig et al., Cell 2017).  We extended this work to reveal the precise binding interface between Rubisco and its multivalent linker EPYC1, then mapped these interactions into the native pyrenoid to produce a molecular model of the liquid pyrenoid matrix (He et al., Nat Plants 2020).  As pyrenoids have independently evolved numerous times, we are exploring pyrenoid organization in a variety of ecologically important marine algae, including diatoms and dinoflagellates.

Centrioles and Cilia
Using Naegleria, Chlamydomonas, Paramecium, and mammalian cells, we collaborated with the Guichard/Hamel group (Univ. Geneva) to explore how centrioles assemble in diverse evolutionary lineages (Le Guennec et al., Science Advances 2020; Klena et al., EMBO Journal 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.  Along these lines, we resolved the native structure of the Chlamydomonas transition zone and observed the stepwise assembly of IFT trains at the ciliary base (van den Hoek et al., bioRxiv 2021).

​Phase-Separated Compartments
In addition to our studies describing ER-associated proteasome clusters and the liquid-like nature of the pyrenoid matrix (both described above), 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). 

Methodology Development
Finally, we strive to improve methods for cryo-ET data acquisition and analysis,  both through partnerships and "in house".  A recent example is our collaboration with the Kervrann group (Inria CNRS) and the Peng group (Helmholtz AI) on DeepFinder software, which uses neural networks to identify molecular complexes within cellular tomograms (Moebel et al., Nat Methods 2021).

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