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, we use focused ion beam (FIB) milling of frozen cells followed by cryo-electron tomography to image macromolecules within their native cellular environment. Through a combination of nanometer-precision localization and high-resolution structural analysis, we aim to chart the molecular landscapes of organelles. 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 are investigating mammalian cells, yeast, several species of marine algae, and the ameoboflagellate Naegleria gruberi.

Zoom Image

Large panel, top left: proteasomes tether to NPCs (purple) at two distinct sites (red: membrane-tethered proteasomes ... [more]

Large panel, top left: proteasomes tether to NPCs (purple) at two distinct sites (red: membrane-tethered proteasomes, yellow: basket-tethered proteasomes, blue: free proteasomes). The nuclear envelope (grey), ribosomes (black/white) and a mitochondrion (red, with yellow ATP synthases) are also shown. Inset: view of an NPC from inside the nucleus. Large panel, bottom left: protein arrays maintain the narrow luminal spacing at the center of the trans-Golgi cisternae. Grey image: XY slice from the tomogram, colored structure: subtomogram average of the array. Large panel, top right: CO₂-fixing rubisco enzymes (blue) are packed within the pyrenoid with liquid-like organization. Pyrenoid tubules (green) and minitubules (yellow) are also shown. Large panel, bottom right: Native architecture of thylakoid membranes (green) within the cell. Thylakoid tips converge at the chloroplast envelope (blue). Starch (tan) and the pyrenoid (grey) are also shown. Bottom row: subtomogram averages of a variety of macromolecules from Chlamydomonas. From left to right: nuclear envelope-tethered proteasome, the nuclear pore complex, ribosome bound to the ER translocon, COPI coat on Golgi vesicles and buds, Rubisco within the pyrenoid. [less]

Large panel, top left: proteasomes tether to NPCs (purple) at two distinct sites (red: membrane-tethered proteasomes, yellow: basket-tethered proteasomes, blue: free proteasomes). The nuclear envelope (grey), ribosomes (black/white) and a mitochondrion (red, with yellow ATP synthases) are also shown. Inset: view of an NPC from inside the nucleus. Large panel, bottom left: protein arrays maintain the narrow luminal spacing at the center of the trans-Golgi cisternae. Grey image: XY slice from the tomogram, colored structure: subtomogram average of the array. Large panel, top right: CO₂-fixing rubisco enzymes (blue) are packed within the pyrenoid with liquid-like organization. Pyrenoid tubules (green) and minitubules (yellow) are also shown. Large panel, bottom right: Native architecture of thylakoid membranes (green) within the cell. Thylakoid tips converge at the chloroplast envelope (blue). Starch (tan) and the pyrenoid (grey) are also shown. Bottom row: subtomogram averages of a variety of macromolecules from Chlamydomonas. From left to right: nuclear envelope-tethered proteasome, the nuclear pore complex, ribosome bound to the ER translocon, COPI coat on Golgi vesicles and buds, Rubisco within the pyrenoid.

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 (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).

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 are currently investigating cytoplasmic regions adjacent to the ER membrane that are concentrated with proteasomes and cdc48, which work together to perform ER-associated degradation.

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).

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 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 interested in exploring pyrenoid organization in other species, including diatoms and dinoflagellates.

Centrioles and Cilia

Using Naegleria, Chlamydomonas, and mammalian cells, we are exploring how centrioles assemble and how the ciliary pore gates the transit of both soluble and membrane proteins between the cell and the cilium.

Phase-Separated Compartments

In addition to our work describing how the pyrenoid is a phase-separated liquid-like 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).

Publications

2019

Schaffer M., Pfeffer S., Mahamid J., Kleindiek S., Laugks T., Albert S., Engel B.D., Rummel A., Smith A.J., Baumeister W., Plitzko J.M.: A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Nature Methods, 16:757–762, 2019  Full Paper

Theis J., Kumar Gupta T., Klingler J., Wan W., Albert S., Keller S., Engel B.D., Schroda M.: VIPP1 rods engulf membranes containing phosphatidylinositol phosphates. Scientific Reports, 9:8725, 2019 Full Paper

Rast A., Schaffer M., Albert S., Wan W., Pfeffer S., Beck F., Plitzko J.M., Nickelsen J., Engel B.D.: Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane. Nature Plants, 5:436-446, 2019 Full Paper Commentary in Molecular Plant

Schuller J.M., Birrell J.A., Tanaka H., Konuma T., Wulfhorst H., Cox N., Schuller S.K., Thiemann J., Lubitz W., Sétif P., Ikegami T., Engel B.D., Kurisu G., Nowaczyk M.M.: Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer. Science, 363:257-260, 2019 Full Paper Commentary in Science RUB Press Release

2018

Kovtun O., Leneva N., Bykov Y.S., Ariotti N., Teasdale R.D., Schaffer M., Engel B.D., Owen D.J., Briggs J.A.B., Collins B.M.: Structure of the membrane-assembled retromer coat by cryo-electron tomography. Nature, 561:561–564, 2018 Full Paper Commentary in Current Biology MRC LMB Press Release Univ. of Queensland Press Release EMBL Press Release 

Delarue M., Brittingham G.P., Pfeffer S., Surovtsev I.V., Pinglay S., Kennedy K.J., Schaffer M., Gutierrez J.I., Sang D., Poterewicz G., Chung J.K., Plitzko J.M., Groves J.T., Jacobs-Wagner C., Engel B.D., Holt L.J.: mTORC1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding. Cell, 174:338-349.e320, 2018. Full Paper Commentary in Cell MPI Press Release Commentary in Nature Chemical Biology  NYU Press Release MBL Press Release

Mosalaganti S., Kosinski J., Albert S., Schaffer M., Strenkert D., Salomé P.A., Merchant S.S., Plitzko J.M., Baumeister W., Engel B.D., Beck M.: In situ architecture of the algal nuclear pore complex. Nature Communications, 9:2361, 2018. Full Paper EMBL Press Release

2017

Albert S., Schaffer M., Beck F., Mosalaganti S., Asano S., Thomas H.F., Plitzko J.M., Beck M., Baumeister W., Engel B.D.: Proteasomes tether to two distinct sites at the nuclear pore complex. PNAS. 114: 13726-13731, 2017. Full Paper  MPI Press Release

Bykov Y.S., Schaffer M., Dodonova S.O., Albert S., Plitzko J.M., Baumeister W., Engel B.D., Briggs J.A.G.: The structure of the COPI coat determined within the cell. eLife. 6:e32493, 2017. Full Paper Commentary in eLife Commentary in Science

Freeman Rosenzweig E.S., Xu B., Kuhn Cuellar L., Martinez-Sanchez A., Schaffer M., Strauss M., Cartwright H.N., Ronceray P., Plitzko J.M., Förster F., Wingreen N.S., Engel B.D., Mackinder L.C.M., Jonikas M.C.: The eukaryotic CO2-concentrating organelle is liquid-like and exhibits dynamic reorganization. Cell. 171:148-162.e19, 2017. Full Paper Commentary in Cell  MPI Press Release  Princeton Press Release

Pfeffer S., Dudek J., Schaffer M., Ng B.G., Albert S., Plitzko J.M., Baumeister W., Zimmermann R., Freeze H.H., Engel B.D., Förster F.: Dissecting the molecular organization of the translocon-associated protein complex. Nature Communications. 8:14516, 2017. Full Paper Utrecht Press Release

Schaffer M., Mahamid J., Engel B.D., Laugks T., Baumeister W., Plitzko J.: Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. Journal of Structural Biology. 197:73-82, 2017. Full Paper

2016

Asano S., Engel B.D., Baumeister W.: In situ cryo-electron tomography: a post-reductionist approach to structural biology. Journal of Molecular Biology. 428: 332–343, 2016. Full Paper

2015

Schaffer M., Engel B.D., Laugks T., Mahamid J., Plitzko J.M., Baumeister W.: Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography. Bio-protocol. 5:e1575, 2015. Full Paper

Engel B.D., Schaffer M., Albert S., Asano S., Plitzko J.M., Baumeister W.: In situ structural analysis of Golgi intracisternal protein arrays. PNAS. 112: 11264-11269, 2015. Full Paper MPI Press Release

Engel B.D., Schaffer M., Cuellar Kuhn L., Villa E., Plitzko J.M., Baumeister W.: Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. eLife. 4: e04889, 2015. Full Paper Commentary in eLife MPI Press Release