Category Archives: Publications

Synergy between cyclase-associated protein and cofilin accelerates actin filament depolymerization by two orders of magnitude

From Science at Brandeis:

“All animal and plant cells contain a highly elaborate system of filamentous protein polymers called the actin cytoskeleton, a scaffold that can be rapidly transformed to alter a cell’s shape and function. A critical step in reconfiguring this scaffold is the rapid disassembly (or turnover) of the actin filaments. But how is this achieved? It has long been known that the protein Cofilin plays a central role in this process, but it has been unclear how Cofilin achieves this feat. Cofilin can sever actin filaments into smaller fragments to promote their disassembly, but whether it also catalyzes subunit dissociation from filament ends has remained uncertain and controversial. Until now, this problem has been difficult to address because of limitations in directly observing Cofilin’s biochemical effects at filament ends….”  Dr. Shashank Shekhar, working together with Dr. Johnson Chung and “jointly mentored by Bruce Goode, Jeff Gelles and Jane Kondev, use[d] microfluidics-assisted single molecule TIRF imaging to tackle the problem.

The new study shows that Cofilin and one other protein (Srv2/CAP) intimately collaborate at one end of the actin filament to accelerate subunit dissociation by over 300-fold! These are the fastest rates of actin depolymerization ever observed. Further, these results establish a new paradigm in which a protein that decorates filament sides (Cofilin) works in concert with a protein that binds to filament ends (Srv2/CAP) to produce an activity that is orders of magnitude stronger than the that of either protein alone.”

10.1038/s41467-019-13268-1
Shekhar S. et al. Synergy between cyclase-associated protein and cofilin accelerates actin filament depolymerization by two orders of magnitude.
Nature Communications
10, 5319 (2019).

A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding

In this project, Kankowan Champasa from Stephen Bell’s lab at MIT collaborated with other researchers from the Bell and Gelles labs to study a key process that sets the stage for replication of chromosomal DNA. They explain “licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally.”  The researchers identified a conserved motif in the Mcm4 helicase subunit essential for formation of productive replication complexes.  Single-molecule fluorescence energy transfer experiments show that mutations in the motif still allow the two hexamers to come into contact, but they prevent the formation of the stable double-hexamers that perform the extensive DNA unwinding needed for replication.

10.7554/eLife.45538
A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding.
Champasa, K., Blank, C., Friedman, L.J., Gelles, J., and Bell, S.P.
eLife (2019) 8:e40576

Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone

Grp94 is a molecular chaperone that helps to fold and maintain the folded state of “client” proteins in the endoplasmic reticulum.  Acceleration of client folding is driven by conformational changes in Grp94.  However,  the sequence of conformational changes and how these changes are coupled to the cycle of ATP hydrolysis is not well understood.  Prof. Timothy Street and his lab members Bin Huang and Ming Sun, in collaboration with Larry Friedman, did single-molecule fluorescence resonance energy transfer (FRET) experiments to directly observe conformational cycling in individual Grp94 molecules.  Their studies show that ATP hydrolysis can drive repeated cycling between alternative “closed” states of Grp94, suggesting a way that enzyme might propagate structural changes to client molecules. Chemical scheme for conformational cycling of Grp94.

10.1016/j.jmb.2019.06.004
Conformational Cycling within the Closed State of Grp94, an Hsp90-Family Chaperone
Huang, B., Friedman, L.J., Gelles, J., Sun, M., and Street, T.O.
Journal of Molecular Biology 431, 3312-3323 (2019).

Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

RNA polymerases contain a conserved “secondary channel” in which proteins that regulate transcription can bind. In Escherichia coli bacteria, the secondary channel factors (SCFs) GreB and DksA both repress initiation of ribosomal RNA synthesis, but SCF loading and repression mechanisms are unclear.  Sarah Stumper and her collaborators used fluorescence correlation spectroscopy and multi-wavelength single-molecule fluorescence colocalization microscopy to show that the SCFs likely repress transcription through an interesting “delayed inhibition” mechanism in which the proteins arrive at DNA already complexed to RNA polymerase and block at a later stage of transcription initiation.  The work explains factors that control the relative contributions of the two proteins to regulation and suggests a mechanism by which repression is restricted to ribosomal RNA and other promoters that form short-duration complexes with RNA polymerase.

10.7554/eLife.40576
Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription.
Stumper, S.K., Ravi, H., Friedman, L.J., Mooney, R.A., Corrêa, I.R., Gershenson, A., Landick, R., and Gelles, J.
eLife (2019) 8:e40576

Abp1 regulation of branched actin networks

Branched actin filament networks formed by the Arp2/3 complex play an essential role in force production in eukaryotic cells.  Branched networks are not static components of the cytoskeleton.  Instead the times and locations of network assembly  and disassembly are tightly controlled by regulatory proteins.  Ph.D. student Siyang Guo used single-molecule fluorescence methods to show how the Abp1 protein positively regulates branched actin networks.  Remarkably, Apb1 functions by two distinct mechanisms.  The protein stimulates the formation of networks by stabilizing the binding of Arp2/3 complex to the sides of actin filaments, a precursor to branch formation.  However after branches form bound Abp1 works differently: it protects the network from GMF, the “pruning shears” protein that chops off branches during network disassembly.  Taken as a whole, the study gives deeper insight into the multiple layers of regulation that control cytoskeleton pattern formation and dynamics.  This project is part of a long-term collaboration on cytoskeletal regulation with Bruce Goode’s lab.

10.1038/s41467-018-05260-y
Abp1 promotes Arp2/3 complex-dependent actin nucleation and stabilizes branch junctions by antagonizing GMF
Siyang Guo, Olga S. Sokolova, Johnson Chung, Shae Padrick, Jeff Gelles, Bruce L. Goode
Nature Communications (2018) 9:2895.

“Synergistic assembly of human pre-spliceosomes across introns and exons”

In human genes, protein-coding regions alternate with non-coding “introns” that must be snipped out of the RNA transcript before it is used to produce a protein. The snipping is done by the spliceosome, a complex molecular machine that must assemble anew on each intron it removes.  The spliceosome must cut out exactly the segments of the messenger molecule that require removal, no more and no less, since inaccurate intron removal can produce a messenger molecule that is non-functional or that causes disease.

Implications of synergistic U2 recruitment for the mechanism of exon and intron recognition.To help understand how multiple introns are quickly and accurately removed, postdoctoral fellow Joerg Braun developed a light microscopy method by which for the first time we can observe the coordinated processes by which human spliceosomes recognize and assemble around the segments of single messenger RNA molecules.  As the eLife digest puts it: “The experiments show that spliceosomes working on different introns in the same pre-mRNA actually help each other out. As one assembles, this helps the spliceosome that processes the neighboring intron to get built. In particular, the U1snRNPs [a spliceosome sub-assembly] processing nearby introns collaborate to promote the assembly and activity of the spliceosomes. This teamwork is likely important to guarantee that multiple introns are cut out quickly and accurately.”

10.7554/eLife.37751
Synergistic assembly of human pre-spliceosomes across introns and exons.
Joerg E. Braun, Larry J. Friedman, Jeff Gelles, and Melissa J. Moore.
eLife (2018) 7:e37751.
Resources:
New plasmids reported in this article can be obtained from Addgene.
Computer software used in this research can be obtained from GitHub.

“Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing”

Mcm2-7 is a ring-shaped DNA helicase that plays an essential role in DNA repliction in eukaryotic cells.  Two of the helicase molecules must encircle the double-stranded DNA at a replication origin, establishing a loaded, anti-parallel double-ring complex able to start replication at the appropriate cell cycle stage.  In this study, Simina Ticau together with collaborators from Steve Bell’s lab (MIT), Jeff Gelles’ lab (Brandeis), and New England BioLabs used wild-type and mutant helicases in single-molecule colocalization (“CoSMoS”) and single-molecule fluorescence resonance energy transfer (smFRET) experiments to identify the mechanisms by which regulatory factors and nucleotide hydrolysis control ring opening and coordinate loading. This work reveals the molecular processes that serve to prevent catastrophic genome damage due to incorrect or mistimed assembly of the replicative machinery.

10.1038/nsmb.3375
Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing
Simina Ticau, Larry J Friedman, Kanokwan Champasa, Ivan R Corrêa Jr, Jeff Gelles, Stephen P Bell
Nat. Struct. Molec. Biol. (2017) 24: 309–315.

Dynamics of GreB-RNA polymerase interaction

In living cells, messenger RNAs are not manufactured by RNA polymerases (RNAPs) functioning alone.  Instead, RNA synthesis is carried out collectively by RNAP together with accessory proteins that associate with the RNAP-containing transcription elongation complex and modulate its activity.  In this paper, Larry Tetone, Larry Friedman, and Melissa Osborne, along with their collaborators from the Gelles and Landick labs, used multi-wavelength single-molecule fluorescence methods to for the first time directly observe the dynamic binding and dissociation of an accessory protein with an RNAP during active transcript elongation.  The protein, GreB, is important for transcript proofreading in E. coli and other bacteria and is a functional analog of the TFIIS protein in eaukaryotes.  “Unexpectedly,” the authors report, “GreB was not selectively recruited to RNAPs requiring its transcript proofreading function. Instead, GreB transiently bound to multiple types of complexes, eventually finding via random search RNAPs that require its activity. The observations suggest a paradigm by which a regulator can act while minimizing obstruction of a binding site that must be shared with other proteins.”

10.1073/pnas.1616525114
Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue
Larry E. Tetone, Larry J. Friedman, Melisa L. Osborne, Harini Ravi, Scotty Kyzer, Sarah K. Stumper, Rachel A. Mooney, Robert Landick, and Jeff Gelles
PNAS (2017) 114:E1081-E1090.

Resources:
New plasmids reported in this article can be obtained from Addgene

“The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers”

The physical forces that drive oligomerization of soluble proteins are well understood and have been extensively studied.  For proteins with transmembrane domains — transport enzymes, for example — oligomerization is often essential for function but its physical basis is less clear.  In this project,  Janice Robertson devised a new method based on liposome extrusion and single-molecule fluorescence photobleaching analysis to accurately measure the dimer association free energy of a ClC-type chloride ion/hydrogen ion antiporter.  (Janice started this work when she was a postdoc in Chris Miller’s lab at Brandeis and later completed the project in her own  lab at the University of Iowa.)  The study reveals that ClC-ec1 “is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.”

10.7554/eLife.17438
The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers
Rahul Chadda, Venkatramanan Krishnamani, Kacey Mersch, Jason Wong, Marley Brimberry, Ankita Chadda, Ludmila Kolmakova-Partensky, Larry J Friedman, Jeff Gelles, and Janice L Robertson
eLife (2016) 5:e17438

“Single molecule analysis reveals reversible and irreversible steps during spliceosome activation”

“The spliceosome is a complex molecular machine, composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins, that excises introns from precursor messenger RNAs (pre-mRNAs).  After assembly, the spliceosome is activated for catalysis by rearrangement of subunits to form an active site.” This study used multi-wavelength single-molecule fluorescence (“CoSMoS”) techniques to elucidate the mechanism of budding yeast spliceosome activation.  Activation turns out to be unexpectedly dynamic and variable: some spliceosomes  take multiple attempts to activate and the pathway contains both reversible and irreversible steps.  Strikingly, ATP powers both steps that drive the process forward toward splicing and well as reverse steps that diassemble intermediates to allow subsequent re-attempts at activation. These findings give new insight into how the efficiency and fidelity of pre-mRNA splicing is maintained.Predominant spliceosome activation pathway

The scientific project in this paper was initiated by Aaron Hoskins during his postdoctoral work in Melissa Moore’s and Jeff Gelles’ labs, but it was brought to fruition by Aaron and Margaret Rodgers working in Aaron’s lab at Univ. Wisconsin, Madison.

10.7554/eLife.14166
Single molecule analysis reveals reversible and irreversible steps during spliceosome activation
Aaron A. Hoskins Margaret L. Rodgers , Larry J. Friedman , Jeff Gelles , Melissa J. Moore
eLife (2016) 5:e14166