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.”
Chung J, et al. Single-molecule analysis of actin filament debranching by cofilin and GMF.
PNAS,119, e2115129119 (2022)
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.
Ordabayev Y.A., et al. Bayesian machine learning analysis of single-molecule fluorescence colocalization images
eLife, 11, e73860 (2022)
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.
Gupta S., et al. A helicase-tethered ORC flip enables bidirectional helicase loading
eLife 10, e74282 (2021)
This article was the subject of an eLife “Insight article” by Bruce Stillman.
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.
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).
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, 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.”
together with collaborators from 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)
DNA transcription by RNA polymerase II (RNApII) is arguably the process most central to regulation of gene expression in eukaryotic organisms. Regulated transcription requires the formation on DNA of molecular assemblies containing not only RNApII but also dozens of accessory proteins that play pivotal roles in the process. While we know about the structures of some of these assemblies in atomic detail, quantitative understanding of the dynamics and pathways by which the assemblies interconvert and progress through this fundamental gene expression pathway is largely lacking.
In this study we report single-molecule fluorescence microscopy studies of transcription in yeast nuclear extract, for the first time visualizing and measuring the dynamics of activator-dependent recruitment of RNApII and the central elongation factor Spt4/5 to transcription complexes. Grace Rosen (Jeff Gelles’ labortatory, Brandeis) , Inwha Baek (Steve, Buratowski’s lab, Harvard Medical School), and collaborators elucidated the kinetically significant steps in activated RNApII transcription initiation and show for the first time that Spt4/5 dynamics are tuned to the typical lifetimes of transcription elongation complexes. In addition to these substantive results, our work represents an important methodological advance. As the first application of the CoSMoS (co-localization single-molecule spectroscopy) technique to activated eukaryotic transcription, it demonstrates a general method for elucidating the correlated dynamic interactions of different components of the machinery with initiation and elongation transcription complexes. The approach is likely to find further use in studies of the mechanistic features of RNApII transcription.
Rosen, G.A., Baek, I., et al., Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
DNA transcription is the most important nexus of gene regulation in all living organisms. In recent years, single-molecule experiments have given us a new window into the mechanisms of transcription and have revealed novel, previously unsuspected molecular behaviors and mechanisms. Ph.D. student Tim Harden used multi-color single-molecule fluorescence imaging to reveal a completely new transcription cycle for bacterial RNA polymerase. Harden and co-authors showed that an RNA polymerase molecule in vitro frequently (in >90% of transcription events) remains bound to DNA and may again initiate transcription after it has terminated the first round of transcription. Even more unexpectedly, this “secondary initiation” is not restricted to the same RNA. After the first round, the polymerase can scan thousands of basepairs along the DNA and can initiate at a different start site, frequently one that is oriented in the opposite direction and produces an antisense transcript.
To complement the single-molecule studies in vitro, the manuscript reports new analyses of whole-transcriptome cellular RNAs revealed by the Rend-seq method that measures transcript initiation and termination frequencies across the genome with single basepair resolution. These provide evidence that the new transcription cycle may be responsible for initiating antisense transcription at hundreds of genomic locations in the two widely divergent bacterial species examined. The work defines a new mechanism for the regulated production of antisense RNAs, many of which are now recognized as important agents of gene-specific regulation through control of transcription, mRNA decay, and translation. In addition, the new transcription cycle provides a mechanism through which transcription initiation can be controlled not just through feedback networks involving multiple genes, but also through production of multiple different primary transcripts consequent to a single RNA polymerase-to-DNA recruitment event.
Harden, T.T., et al. Alternative transcription cycle for bacterial RNA polymerase.
Nature Communications 11, 450 (2020).
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).
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
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