Author Archives: Lab

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.”

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.

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.

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).

Grace Rosen, Ph.D.

Congratulations to Grace Rosen on completing her Ph.D. defense!  Grace will be working post-graduation as a scientist at the Boston VA Medical Center.

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.

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

Kinosita Award

Jeff received the 2019 Kazuhiko Kinosita Award in Single-Molecule Biophysics from the Biophysical Society.  The award is named after Prof. Kazuhiko Kinosita, Jr. who was a much-admired pioneer of single-molecule biophysics, famous for his creative and intellectually rigorous approach to science. His research revealed key features of how molecular motors operate and how cells make ATP.  Students will enjoy this public lecture from the January 2015 Single Molecule Biophysics conference in which Prof. Kinosita talks about his work:

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.

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.