Research Overview

My colleagues and I discovered and characterized proteins that produce forces for cellular movements. We led the field toward a deep understanding of the molecular mechanisms of cellular movements and cytokinesis. 

1. Discovery and characterization of cytoplasmic contractile proteins

As a medical student I used an in vitro motility system to provide the first direct link between cytoplasmic actin filaments and cellular movements. My discovery of the first unconventional myosin (myosin-I) as a postdoc launched the search for the diversity of molecular motors. We also linked myosin-I to organelle movements and endocytosis.

• Pollard TD, Korn ED (1973) Acanthamoeba myosin I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J. Biol. Chem. 248:4682-4690. PMID: 4268863. (First unconventional myosin.)

• Adams RJ, Pollard TD (1986) Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature 322:754-756. PMID: 3748157 (Discovery of myosin-based organelle movements)

We have done the definitive work on the mechanism of actin polymerization including the kinetic constants for nucleation, elongation and ATP hydrolysis and the effects of the bound nucleotide on these reactions. This quantitative data is the foundation upon which all studies of actin polymerization depend. We used cryo-EM to determine the first high resolution (3.1 Å) structures of actin filaments with bound AMPPNP, ADP-Pi and ADP, which answered decades of questions about the mechanism of polymerization. Collaborative molecular dynamics simulations with Greg Voth’s lab revealed the mechanisms of subunit addition at the barbed end and of phosphate release from ADP-Pi-actin filaments.

• Pollard TD (1986) Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J. Cell Biol. 103:2747-2754. PMID: 3793756 (Rate constants for actin filament elongation and disassembly)

• Chou S, Pollard TD (2019) Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides. Proc. Nat. Acad. Sci. USA. 116:4265-4274. PMID: 30760599.

• Wang, Y., Wu, J., Zsolnay, V., Pollard, T.D. and Voth, G.A. (2024) Mechanism of Phosphate Release from Actin Filaments. Proc. Nat. Acad. Sci. USA. 121:e2408156121. PMID: 38980907.

Postdoc Gerhard Isenberg discovered capping protein, which we and my student John Cooper subsequently characterized in detail. We also characterized the mechanisms and determined the atomic structures of two important actin monomer-binding proteins, profilin and cofilin.

2. Discovery and characterization of Arp2/3 complex

Graduate student Laura Machesky discovered Arp2/3 complex. In collaboration with Machesky we discovered that the Wiskott-Aldrich syndrome protein (WASp) and related nucleation-promoting factors stimulate Arp2/3 complex to nucleate actin polymerization.

• Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J, Pollard TD (1994) A cortical complex of seven Acanthamoebapolypeptides including two unconventional actins binds to profilin. J. Cell Biol. 127:107-115. PMID: 7929556 (Discovery of Arp2/3 complex and its localization at the leading edge of motile cells)

• Machesky LM, Mullins RD, Higgs HN, Kaiser DA, Blanchoin L, May RC, Hall ME, Pollard TD (1999) WASp-related protein Scar activates dendritic nucleation of actin filaments by Arp2/3 complex. Proc. Nat. Acad Sci. USA 96: 3739-3744. PMID: 10097107 (Discovery that WASp-Scar proteins activate Arp2/3 complex)

We determined the first high resolution structure of Arp2/3 complex followed by crystal structures of Arp2/3 complex with various bound nucleotides and the first drug-like inhibitor, CK666, which is now widely used in cell biology experiments.

• Robinson RC, Turbedsky K, Kaiser DA, Higgs HN, Marchand J-B, Choe S, Pollard TD (2001) Crystal structure of Arp2/3 complex. Science 294:1679-1684. PMID: 11721045 (High resolution crystal structure of Arp2/3 complex with a proposal for the mechanism of branch formation)

We characterized the pathway whereby Arp2/3 complex, nucleation-promoting factors such as WASp, actin filaments and actin monomers form branched actin filaments that generate forces for cellular motility. Our high resolution cryo-EM structure branch junction revealed the pathway of branch formation.

• Espinoza S, Metskas LA, Chou SZ, Rhoades E, Pollard TD (2018) Conformational changes in Arp2/3 complex induced by WASp-VCA and actin filaments. Proc Nat Acad Sci USA 115:E8642-E8651. PMID: 3015041

• Chou, S.Z., Chatterjee, M. and Pollard. T.D. (2022) Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high resolution cryo-EM structure of the branch junction. Proc. Nat. Acad. Sci. USA. 119:e2206722119. PMID: 36442092.

3. Development and testing of molecular models for cellular motility

Motility uber figure

Our work on actin, profilin, cofilin, capping protein and Arp2/3 complex led to the highly influential “dendritic nucleation hypothesis” that explains how branched actin filaments produce the forces for cellular movements.

• Mullins RD, Heuser JA, Pollard TD (1998) The interaction of the Arp2/3 complex with actin:  nucleation, high affinity pointed end capping and formation of branching networks of filaments. Proc. Nat. Acad. Sci. USA 95:6181-6186. PMID: 9600938 (Discovery of actin filament branch formation by Arp2/3 complex inspired the dendritic nucleation hypothesis for cellular motility)

By combining quantitative light microscopy and mathematical modeling we verified the central features of the dendritic nucleation hypothesis as it operates in live yeast cells during clathrin-mediated endocytosis. 

• Berro J, Pollard TD (2014) Local and global analysis of endocytic patches dynamics in fission yeast using a new “temporal super-resolution” realignment method. Molec. Biol. Cell. 25:3501-3514. PMID: 25143395

• Arasada R, Sayyad WA, Berro J, Pollard TD (2018) High-speed super-resolution imaging of the organization of the proteins in fission yeast clathrin-mediated endocytic actin patches. Molec Biol Cell 29:295-303. PMID: 29212877.

Importance of this work: The highly conserved genes for the actin system evolved more than a billion years ago, so our discoveries in model systems revealed general principles relevant to human cells. Understanding the mechanism of cellular motility is important, because cellular movements powered by actin polymerization are required for leukocytes to capture pathogens and embryonic development including laying down one million miles of connections between the cells in the human brain. On the darker side, movements of malignant cells from primary tumors to secondary sites are the leading cause of death in cancer patients.

4. Characterization of cytokinesis proteins

In 1976 Postdoc Keigi Fujiwara discovered myosin-II concentrates in the cytokinetic cleavage furrow of Hela cells, key evidence for the concept that a contractile ring of actin filaments and myosin pinches cells in two during cytokinesis. During the 1980s we characterized in detail the assembly of cytoplasmic myosin-II filaments and the ultrastructure of the contractile ring by electron microscopy of thin sections.

Starting in the late 1990s, we turned to fission yeast to study the molecular pathway of contractile ring assembly, constriction and disassembly to take advantage of the excellent inventory of cytokinesis genes compiled by geneticists. We purified from fission yeast and characterized important contractile ring proteins (actin, formins, profilin, cofilin, two isoforms of myosin-II, anillin, two F-BAR proteins and IQGAP). This work included the most detailed analysis of how formins nucleate and elongate actin filaments, while associated processively with the growing barbed end of the actin filament.

• Kovar DR, Harris ES, Mahaffy RE, Higgs HN, Pollard TD (2006) Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 724:423-435. PMID: 16439214 (Mechanism of actin assembly by formins)

CK timeline horizontal b

Postdoc Jian-Qiu Wu determined with high precision the time course of events during cytokinesis and developed methods to count protein molecules in live cells by fluorescence microscopy, measuring the numbers and dynamics of more than 30 contractile ring proteins. We used quantitative fluorescence microscopy to discover that actin filaments shorten as the contractile ring constricts and to show that three myosins contribute to contractile ring assembly and constriction. 

• Wu J-Q, Kuhn JR, Kovar DR, Pollard TD (2003) Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Devel. Cell 5:723-734. PMID: 14602073 (This study established the molecular road map for assembly of the cytokinetic contractile ring, which set our experiment agenda ever since.)

We used high-speed super-resolution fluorescence microscopy of live cells to determine the organization of the precursors of the contractile ring. This work showed that nodes are uniform structures and mapped the locations of five of the node proteins with 35 nm resolution. These insights were the inspiration for the second generation of models of contractile ring constriction.

• Laplante C, Huang F, Tebbs IR, Bewersdorf J, Pollard TD. (2016) Molecular organization of cytokinesis nodes and contractile ring by super- resolution fluorescence microscopy of live fission yeast. Proc. Nat. Acad. Sci. USA. 113:E5876-E5885. PMID: 27647921.

5. Development and testing of molecular models for cytokinesis

Our body of quantitative data on fission yeast cytokinesis proteins and the events in live cells allowed us to formulate and simulate molecularly explicit mathematical models for contractile ring assembly and constriction. These models were the first to account accurately for the physical steps in cytokinesis and provide a framework to study the biochemical systems that regulate the transitions in the system during the cell cycle.

• Vavylonis D, Wu J-Q, Hao S, O’Shaughnessy B, Pollard TD (2008) Assembly mechanism of the contractile ring for cytokinesis by fission yeast. Science 319:97-100. PMID: 18155236 (A combination of experimental measurements on live cells and simulations of a mathematical model tested the most advanced hypothesis for the assembly of the contractile ring.)

• Chen Q, Pollard TD (2011) Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring. J. Cell Biol. 195:485-498. PMID: 22024167 (Experiments & modeling revealed cofilin is responsible for the release step in the search-capture-pull-release model.)

• Akamatsu MS, Berro J, Pu K-M, Tebbs IR, Pollard TD (2014) Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase. J. Cell Biol. 204:977-988. PMID: 24637325 (Experiments and modeling to characterize the “node cycle” around the entire cell cycle) 

• Stachowiak MR, Laplante C, Chin HF, Guirao B, Karatekin E, Pollard TD, O’Shaughnessy B (2014) Mechanism of cytokinetic ring constriction in fission yeast. Devel. Cell 29:547-561. PMID: 24914559. (These simulations of a molecularly explicit model are the first to produce the tension observed in the contractile ring and explain why rapid turnover of contractile ring proteins is essential for ring constriction.)

Importance of this work: Understanding the mechanism of cytokinesis is valuable, because it is the final step of the cell cycle and is required for the equal partition of chromosomes between the daughter cells. Failure of cytokinesis is one cause of aneuploidy during the progression of malignancies. During embryonic development the patterns of cytokinesis shape many tissues. Asymmetric cell divisions are fundamental to stem cell renewal and the development of oocytes.