Author Archives: Lab

Single-molecule analysis of actin filament debranching by cofilin and GMF

The Arp2/3 complex is a multi-component molecular machine that nucleates branched actin filament networks at the leading edge of cells to promote protrusion and at sites of endocytosis to drive membrane invagination. While the process of branched actin nucleation is now well understood (including at mechanistic and structural levels), what is less well understood is how the actin networks are subsequently debranched, or ‘pruned’. Debranching is an absolutely essential step in network remodeling and turnover, which is required for cell motility and endocytosis. The branched actin structures produced by Arp2/3 complex are kinetically stable, with spontaneous dissociation occurring only after tens of minutes to hours, whereas in vivo the branches dissociate in seconds. How is this achieved?

Two separate members of the larger ADF-homology family of proteins, glia maturation factor (GMF) and cofilin, have been implicated in promoting debranching. In this paper, Gelles lab member Johnson Chung, in collaboration with Jeff and with Bruce Goode from the Brandeis Biology Dept., used multi-wavelength single molecule florescence microscopy and quantitative kinetic analysis to define the mechanisms by which these proteins promote debranching.   Dr. Chung shows that “cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch–junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin–actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament–Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.”

10.1073/pnas.2115129119
Chung J, et al. Single-molecule analysis of actin filament debranching by cofilin and GMF.
PNAS,119, e2115129119 (2022)

Bayesian machine learning analysis of single-molecule fluorescence colocalization images

From Science at Brandeis: “Yerdos Ordabayev et al. in the Department of Biochemistry use Bayesian probabilistic programming to implement computer software “Tapqir” for analysis of colocalization single-molecule spectroscopy (CoSMoS) image data. CoSMoS is a tool widely used in vitro to study the biochemical and physical mechanisms of the protein and nucleic acid macromolecular “machines” that perform essential biological functions. In this method, formation and/or dissociation of molecular complexes is observed by single-molecule fluorescence microscopy as the colocalization of binder and target macromolecules each labeled with a different color of fluorescent dye. Despite the use of the method for over twenty years, reliable analysis of CoSMoS data remains a significant challenge to the effective and more widespread use of the technique.

This work describes a holistic causal probabilistic model of CoSMoS image data formation. This model is physics-based and includes realistic shot noise in fluorescent spots, camera noise, the size and shape of spots, and the presence of both specific and nonspecific binder molecules in the images. Most importantly, instead of yielding a binary spot-/no-spot determination, the algorithm calculates the probability of a colocalization event. Unlike alternative approaches, Tapqir does not require subjective threshold settings of parameters so they can be used effectively and accurately by non-expert analysts. The program is implemented in the state-of-the-art Python-based probabilistic programming language Pyro (open-sourced by Uber AI Labs in 2017), which enables efficient use of graphics processing unit (GPU)-based hardware for rapid parallel processing of data and facilitates future modifications to the model. Tapqir is free, open-source software. We envision that [the] program is likely to be adopted by researchers who use single-molecule colocalization methods to study a wide range of different biological systems.”

Yerdos is a postdoctoral fellow jointly advised by Profs. Douglas Theobald and Jeff Gelles.

 

10.7554/eLife.73860
Ordabayev Y.A., et al. Bayesian machine learning analysis of single-molecule fluorescence colocalization images
eLife, 11, e73860 (2022)

Congratulations to Debora!

Congratulations to Dr. Deborah Tenenbaum, shown here with her two Ph.D. mentors Jeff Gelles (smiling under the mask) and Jane Kondev. Debi just successfully defended her combined theory and experiment Physics Ph.D.  thesis.  She will be starting a new position as a postdoc with Justin Kinney at Cold Spring Harbor Laboratory.

The endoplasmic reticulum chaperone BiP is a closure-accelerating cochaperone of Grp94

From the article: “The endoplasmic reticulum (ER) is the site at which secreted proteins (such as the hormone insulin) and membrane-bound proteins are folded. ATP-dependent chaperones within the ER help proteins fold. This study describes how two key ER chaperones, BiP and Grp94, work together at a molecular level. BiP binds to Grp94, which enables Grp94 to change conformation and hydrolyze ATP. In short, BiP provides a signal to switch on Grp94 conformational changes that are required to help other proteins fold. This finding helps explain how two chaperones can work together collaboratively in protein folding. Because BiP and Grp94 are members of highly conserved chaperone families, these findings may provide insight into chaperone-assisted protein folding beyond the ER.”  This project was a collaboration with members of Timothy Street‘s lab in the Brandeis Biochemistry Department.

10.1073/pnas.2118793119
Huang B., et al., The endoplasmic reticulum chaperone BiP is a closure-accelerating cochaperone of Grp94.
PNAS 119, e2118793119 (2022)

Engineering stability, longevity, and miscibility of microtubule-based active fluids

This study describes the formulation and properties of active fluids that contain mixtures of microtubules, polymer solutions, microtubule motor proteins, and ATP. These fluids are isotropic materials that display spontaneous self-organized flow patterns, sometimes persisting over hours, that are a consequence of motor-driven microtubule sliding and polymer-induced microtubule bundling. The project, organized by Zvonimir Dogic’s lab, was a multi-institutional collaboration involving labs from UC Santa Barbara, Hampton Univ., Worcester Polytechnic Inst., Harvard, and Brandeis.

10.1039/D1SM01289D
Chandrakar P., et al., Engineering stability, longevity, and miscibility of microtubule-based active fluids.
Soft Matter 10.1039/D1SM01289D (2022)

A helicase-tethered ORC flip enables bidirectional helicase loading

A key event in eukaryotic DNA replication is origin licensing in G1-phase, during which two Mcm2-7 replicative DNA helicases are loaded onto each origin DNA in an inactive, head-to-head fashion. Origin licensing marks every potential origin in a cell, and the opposing orientation of the loaded helicases ensures that they are poised to initiate bidirectional replication when the cell enters S-phase. Although it has long been known that the origin-recognition complex (ORC) binds origin DNA to direct helicase loading, the molecular mechanism by which two oppositely oriented helicases are loaded remains puzzling. Previous biochemical studies found evidence in support of a two-ORC mechanism for helicase loading wherein each of the two Mcm2-7 helicases are recruited by a separate, oppositely oriented ORC molecule. In contrast, single-molecule and cryo-EM approaches observed predominantly one ORC involved in helicase loading, but could not explain how a single ORC could load two oppositely oriented helicases.

In this paper, a collaboration with Steve Bell’s lab at MIT, Ph.D. student Shalini Gupta reconciles these seemingly contradictory observations. Using single-molecule fluorescence energy transfer (sm-FRET), she observed interactions in vitro between individual ORC molecules and the Mcm2-7 helicases in real time at two separate interfaces. In the large majority of instances, a single ORC molecule recruits both Mcm2-7 helicases through direct interactions. Between recruitment of the first and the second helicase, ORC ‘flips’ its orientation on DNA using a flexible protein tether to the first loaded Mcm2-7. This remarkable ORC inversion ensures that the two helicases are recruited via similar interactions, but in opposite orientations. The data define a complete, integrated pathway for helicase loading that resolves the apparent contradictions between previous observations. The tethered-flip mechanism provides a molecular explanation for how cells avoid the potentially damaging consequences of incompletely-formed helicase pairs at origins.

10.7554/eLife.74282
Gupta S., et al. A helicase-tethered ORC flip enables bidirectional helicase loading
eLife 10, e74282 (2021)

This article was the subject of an eLifeInsight article” by Bruce Stillman.

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

A key event leading to the synthesis of a eukaryotic messenger RNA  is the assembly of a pre-initiation complex (PIC) on promoter DNA near the transcription start site.  The PIC contains RNA polymerase II (pol II) plus general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.  PIC assembly is enhanced by binding of transcription activator proteins to separate DNA sites called upstream activating sequences (UAS) or enhancers, but the dynamic mechanisms by which activators at other sites control PIC assembly has been unclear.  In this paper, Inwha Baek (from the Buratowski lab) and Larry Friedman (from the Gelles lab) defined this mechanism using multi-wavelength single-molecule fluorescence microscopy.  The experiments used budding yeast nuclear extract with fluorescently labeled proteins and the strong artificial activator protein Gal4-VP16.  The investigators found that, unexpectedly, pol II and often TFIIE and TFIIF were not recruited directly to the promoter.  Instead they first bound via the activator to the UAS and were then subsequently transferred, likely as a pre-formed complex, to the promoter.  This work gives new insight into how messenger RNA synthesis is regulated to switch genes on and off in eukaryotic cells.  It also suggests how multiple pol II molecules may be poised at UAS sequences ready to transcribe an adjacent gene, which may explain some of the “bursts” of transcription detected in living cells.

10.1016/j.molcel.2021.07.025
Baek I., et al., Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes
Molecular Cell 81, 3576-3588.e6 (2021).

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

Schematic representation of DNA replication initiation Replication of chromosomal DNA in eukaryotes has two major stages.  Starting in the G1 phase of the cell cycle, double hexamers consisting of two copies of the Mcm2-7 replicative helicase are assembled at replication origins.   Later, in S phase, the two helicases are incorporated into two oppositely oriented CMG (Cdc45-Mcm2-7-GINS) complexes that each then form the core of a replisome.  Control of this “activation” step, which is triggered by the protein kinases DDK and S-CDK, is essential to ensure that each part of the genome is replicated once and only once in each cell cycle.

In this paper, Lorraine De Jesús-Kim together with collaborators from Steve Bell’s and Jeff Gelles’ labs used multi-wavelength single-molecule fluorescence colocation  (“CoSMoS”) methods to study in vitro the molecular mechanism of the activation process.  The journal’s acceptance summary notes that “The manuscript provides new and convincing evidence that a heretofore unknown intermediate state [called “CtG”] for replication start contains multiple copies of the GINS and Cdc45 proteins prior to initiation at each origin with one double hexamer of the MCM2-7 complex. The number of GINS and Cdc45 is determined by DDK phosphorylation of the MCM’s and the probability to create an active helicase (CMG) is increased with multiple numbers of the bound ancillary factors…. The single molecule studies and biochemistry are beautifully executed providing the evidence for such intermediates…. The addition of in vivo studies demonstrates that modulating the multiplicity of DDK phosphorylation (and proposed, CtG formation) has an impact on origin usage in cells.”Proposed model for Cdc45-Mcm2-7-GINS (CMG) formation

10.7554/eLife.65471
Kim L.D.J., et al., DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.
eLife 10, e65471 (2021)