Mir Lab @mirlab.bsky.social · 07/06/2026Thanks! SPARK is parameter-driven, not TF-specific, so it's applicable to any DNA-binding protein with measurable diffusion, binding-site, and residence-time data. As a null model, where kinetics alone can't reproduce the clustering is where higher-order mechanisms may be at play. 140
Mir Lab @mirlab.bsky.social · 07/06/20268/🧵 SPARK was developed by @pennengineering.bsky.social bioengineering graduate student Manya Kapoor @manyakapoor.bsky.social and inspired by our recent experimental results on Pol II (www.nature.com/articles/s41...) and Dorsal clustering (www.biorxiv.org/content/10.1...) 070
Mir Lab @mirlab.bsky.social · 07/06/20267/🧵 We hope SPARK will allow users to assess whether the clusters they observe can be explained by DNA binding and diffusion kinetics, rather than higher-order phenomena like condensate formation. 120
Mir Lab @mirlab.bsky.social · 07/06/20266/🧵 SPARK is packaged with a user-friendly GUI for those who want to gain intuition about how cluster properties emerge and also for use with high-performance computing for more involved simulations and parameter sweeps. Download software and user manual here: doi.org/10.5281/zeno... 110
Mir Lab @mirlab.bsky.social · 07/06/20265/🧵 To demonstrate that simulated data can recapitulate key emergent condensate properties we show that clusters arising from DNA binding can show fluorescence recovery after photobleaching at similar timescales as those reported for condensates. We also observe cluster fusion in simulated movies. 230
Mir Lab @mirlab.bsky.social · 07/06/20264/🧵 In addition to simulating 3D time lapse movies, SPARK can also be used to render super-resolution data for comparison to published work. Image below shows a simulation of Sox2 clustering in mESCs 130
Mir Lab @mirlab.bsky.social · 07/06/20263/🧵 SPARK recapitulates the clustering of transcription factors like Dorsal and Bicoid in Drosophila embryos, and Sox2 in mouse ES cells, despite explicitly excluding protein-protein interactions. (Dorsal clusters shown below) 220
Mir Lab @mirlab.bsky.social · 07/06/20262/🧵 SPARK (Simulation of Protein Accumulation from Reaction Kinetics) takes in experimentally-measurable binding site information and molecular kinetic parameters and generates molecular trajectories. It outputs simulated microscope images under user-defined imaging conditions (mag, exposure, etc.) 251
Mir Lab @mirlab.bsky.social · 07/06/20261/🧵 Can transcription factor condensate formation be explained without phase separation? Our new preprint introduces SPARK, a simulation tool that reproduces condensate behavior (clustering, fusion, FRAP) from diffusion & binding kinetics alone. Movie: 60 sec FRAP sim www.biorxiv.org/content/10.6... 37836
Mir Lab @mirlab.bsky.social · 16/04/2026Very proud of Samantha "S" Fallacaro the first student from our lab to defend their thesis! Check out her latest work (tinyurl.com/42hdfssz) where she shows that "hubs" formed by the transcription factor Dorsal are emergent features of binding kinetics, not higher-order regulatory assemblies. 172
Reposted by Mir LabCAMB Graduate Group @cambupenn.bsky.social · 13/04/2026Samantha Fallacaro in the @mirlab.bsky.social defends her thesis tomorrow! 082
Mir Lab @mirlab.bsky.social · 20/03/2026@mukherja.bsky.social work on RNAPII clusters is in print today: www.nature.com/articles/s41... Congrats! TLDR: RNAPII clusters represent transcriptonally engaged molecules at single genes, not super-stochiometric assemblies. Please check it out and see the the thread below for a summary.nature.comA single cluster of RNA Polymerase II molecules is stably associated with active genes - Nature CommunicationsLive molecular-scale imaging in fly embryos shows that a single RNA Polymerase II cluster stays bound to active genes during transcription. Cluster formation requires initiation, and intensity represe... 04917
Mir Lab @mirlab.bsky.social · 16/01/2026Unless I misunderstood and you are asking about spatially proximal genes in general. In which case it could either, i.e. regulated (for example same TAD) or incidental. 110
Mir Lab @mirlab.bsky.social · 16/01/2026These are sister chromatids (copies of the same chromosome after replication). They are held together by cohesin and other proteins complexes so the proximity is not accidental. if you image with enough temporal and spatial resolution you can incidentally see them separate some times. 120
Mir Lab @mirlab.bsky.social · 16/01/2026One big take away (that took some while for us to reconcile here) is to not assume when you see a bright focus in a cell that a non-stochiometric phenomenon is occurring. 150
Mir Lab @mirlab.bsky.social · 16/01/20264) It is possible that the clusters we observe contain multiple genes that are firing in sync, but our simulations suggest that this is likely rare and the fact that we don’t see thousands of “clusters” is due to differences in Pol II occupancy at genes and technological limitations. 130
Mir Lab @mirlab.bsky.social · 16/01/20263) One observation that was quite convincing to us, is that when we look at the same locus firing on sister chromatids, when they transiently separate, we clearly see two Pol II clusters. By cross linking capture methods these would show a high contact frequency, and by static FISH high overlap. 150
Mir Lab @mirlab.bsky.social · 16/01/20262) When looking at fixed cell images with diffraction limited resolution, there might be multiple clusters at each FISH spot which are not resolvable. 130
Mir Lab @mirlab.bsky.social · 16/01/2026A few points: 1) When interpreting evidence from chromatin conformation methods it is important to keep in mind that live imaging work has now shown that these contacts tend to be quite transient and infrequent in live cells. 120
Mir Lab @mirlab.bsky.social · 05/01/2026We are happy to have contributed single-molecule tracking and super-resolution imaging to this great work from Nik Sgourakis' lab. HLA-Shuttle restores antigen presentation in immunologically cold tumors enabling the identification of new targets for immunotherapy. www.science.org/doi/10.1126/...science.orgHLA-Shuttle: A system for enhancing antigen presentation in immunologically cold tumorsHLA-Shuttle enhances production of HLA class I molecules in neuroblastoma, leading to the discovery of tumor-associated antigens. 040
Reposted by Mir LabApratim (Appy) Mukherjee @mukherja.bsky.social · 18/09/2025Huge props to our grad student @manyakapoor.bsky.social for developing the simulations and the new MS2 analysis amongst other things! We also thank the transcription community for their invaluable feedback and always welcome further discussion😊 032
Reposted by Mir LabAndrew Modzelewski @themodzlab.bsky.social · 13/05/2025WE ARE HIRING! The lab got the Packard AND an R35! Please spread this PostDoc Ad around! Philly and Upenn are a great place to live and work! 2137
Mir Lab @mirlab.bsky.social · 11/04/20255/n: Please reach out directly to me if you are interested. This is a unique opportunity at a time where increased centralized resources are critical to maintain and accelerate the pace of scientific discovery. 010
Mir Lab @mirlab.bsky.social · 11/04/20254/n We are seeking a microscope specialist/engineer. The role will include developing and implementing new microscope hardware, control software, customized analysis pipelines as well as collaborating with core users. 100
Mir Lab @mirlab.bsky.social · 11/04/20253/n We will provide custom built ad hoc optical microscopy solutions, advanced image analysis, and consultation services to researchers across campus and beyond. We will implement technology in response to experimental needs instead of working within the bounds of commercially available technology.med.upenn.edu Advanced Core for Microscope Engineering (ACME) | Advanced Core for Microscope Engineering | Perelman School of Medicine at the University of Pennsylvania 100
Mir Lab @mirlab.bsky.social · 11/04/20252/n: ACME is co-directed by myself (Mustafa Mir), Melike Lakadamyali @melikel.bsky.social (www.lakadamyali-lab.com), Yihui Shen (www.imaging-systems-metabolism-lab.com), and Andrea Stout @aleestoutphd.bsky.social (www.med.upenn.edu/cdbmicroscopycore) 100
Mir Lab @mirlab.bsky.social · 11/04/20251/n We have an exciting new initiative at UPenn to provide custom advanced microscopy solutions: Advanced Core for Microscope Engineering (ACME): www.med.upenn.edu/cdbacme/ We are looking for a specialist to help run this new facility, job description is attached please help share widely! 1109
Mir Lab @mirlab.bsky.social · 09/04/20259/ We welcome feedback, questions, and critical discussion from the community. Huge thanks to all our amazing co-authors for their insight, support, and collaboration throughout this work. 010
Mir Lab @mirlab.bsky.social · 09/04/20258/n: We suggest that enhancers encode the biophysical properties of transcription factor hubs (intensity and stability) through binding affinities and cofactors. Hub properties dictate gene occupancy and transcriptional kinetics, suggesting hubs are active executors of enhancer-encoded regulation. 110
Mir Lab @mirlab.bsky.social · 09/04/20257/n: Hub intensity and duration correlate with burst amplitude, loading rate, and output suggesting that persistently interacting hubs with more molecules tune gene expression. Consistent with the idea that hubs facilitate transcription factor binding and promote expression. 120
Mir Lab @mirlab.bsky.social · 09/04/20256/n: Hub presence at a gene is correlated with increased transcription burst amplitude, Pol II loading rate, and total output. Surprisingly, transient interactions between Dorsal hubs and hunchback (lacking Dorsal binding) boosts transcription in ventral nuclei where Dorsal concentration is highest 120
Mir Lab @mirlab.bsky.social · 09/04/20255/n: We found: • snail has persistent hub interactions with high-intensity hubs • sog: has slightly less persistent hub interactions with lower-intensity hubs • hunchback (negative control): transient interactions without stable hub formation 110
Mir Lab @mirlab.bsky.social · 09/04/20254/n: Next we examined Dorsal hub interactions at two target genes with distinct Dorsal occupancy and enhancer grammars, snai and sog, and as a negative control examined hunchback which has no dorsal sites. 110
Mir Lab @mirlab.bsky.social · 09/04/20253/n: First, we find that hubs form rapidly after mitosis and last through interphase. Nuclear hub density doesn't depend on nuclear concentration but hub intensity does, suggesting that hub formation may be independent of concentration, but hubs accumulate more protein with increased concentrations. 110
Mir Lab @mirlab.bsky.social · 09/04/20252/n: Transcription factor hubs (dense local accumulations) regulate gene expression by promoting the frequency of binding. They are often discussed as having uniform properties based on protein sequence and concentration. We wondered if their biophysical properties vary in a gene dependent manner. 140
Mir Lab @mirlab.bsky.social · 09/04/20251/n: Excited to share our new preprint. We find that Dorsal transcription factor hubs exhibit gene-specific biophysical properties that tune transcriptional output. Read here: www.biorxiv.org/content/10.1.... Project was led by amazing graduate student Samantha Fallacaro (not on bsky)biorxiv.orgTranscription factor hubs exhibit gene-specific properties that tune expressionThe spatial and temporal control of gene expression relies on transcription factors binding to and occupying their target sites. Transcription factor hubs - localized, high-concentration microenvironm... 1129
Reposted by Mir LabNadine Vastenhouw @nvastenhouw.bsky.social · 28/03/2025Martino Ugolini and I wrote a review on how useful embryos are to understand the role of transcription bodies in gene expression. I am sure @mirlab.bsky.social, @harrisonflylab.bsky.social, @lennarthilbert.bsky.social and others would agree. Enjoy the read! portlandpress.com/biochemsoctr....portlandpress.comThe role of transcription bodies in gene expression: what embryos teach usTranscription does not occur diffusely throughout the nucleus but is concentrated in specific areas. Areas of accumulated transcriptional machinery have been called clusters, hubs, or condensates, whi... 2296
Reposted by Mir LabPatrick S. Mitchell @psmitchej.bsky.social · 21/02/2025In light of policies to defund biomedical research, and with judicial hearings happening today, we hope to highlight the many ways biomedical research improves people's lives, and to convey how damaging the proposed cuts would be... not just for us researchers, but for everyone. 23417
Mir Lab @mirlab.bsky.social · 12/02/2025And all the MS2 interaction analysis was done by an amazing bioengineering graduate student @manyakapoor.bsky.social who just joined Bsky! 010
Mir Lab @mirlab.bsky.social · 12/02/2025Major oversight in my earlier postdoc: This work was led by our wonderful postdoc @mukherja.bsky.social who also built the microscope that enabled these experiments 110
Mir Lab @mirlab.bsky.social · 12/02/2025We can't follow the same cluster but see a shift in cluster kinetics across nuclei. We can't say anything about initiation factors since we are just looking at pol2 and we are testing effects of recruitment vs elongation with the drugs. Will check our language to make sure this is clear. Thanks. 010
Mir Lab @mirlab.bsky.social · 12/02/20257/n Overall we show that Pol II clusters are functionally labile and suggest that they likely represent collections of molecules engaged at a single gene locus. Please take a read and send us your critical feedback! 460
Mir Lab @mirlab.bsky.social · 12/02/20256/n Using simultaneous imaging of nascent transcription and Pol II clusters we found that a single cluster remains associated with an active gene during a transcription burst and the intensity of the cluster is highly correlated in real time with the amount of nascent transcription. 130
Mir Lab @mirlab.bsky.social · 12/02/20255/n Using inhibitors of initiation and elongation we found that cluster formation is dependent on initiation and that transcription elongation leads to a reduction in cluster lifetime. 140
Mir Lab @mirlab.bsky.social · 12/02/20254/n We found that Pol II clusters undergo a transformation as the zygotic genome is activated. From initially being mostly composed of molecules engaged in transcription initiation to becoming hotspots of elongating molecules. 130
Mir Lab @mirlab.bsky.social · 12/02/20253/n We used single-molecule tracking and lattice light-sheet microscopy in live Drosophila embryos to ask how clusters change during zygotic genome activation (when there is a ~3x increase in transcription) and how clusters influence transcription levels. 171
Mir Lab @mirlab.bsky.social · 12/02/20252/n Since their discovery in the 1990's the functional roles and mechanisms of formation of RNAPII clusters have been heavily investigated. Models have ranged from transcription factories containing many genes to condensates that transiently "kiss” active genes. 140
Mir Lab @mirlab.bsky.social · 12/02/20251/n Excited to share our new preprint! Using live imaging in Drosophila embryos, we show that RNA Pol II clusters switch from sites of initiation to elongation during zygotic genome activation and that they are stably associated with an active gene: www.biorxiv.org/content/10.1... 59932
Mir Lab @mirlab.bsky.social · 17/01/2025Big congratulations to our MD/PHD student @driaadigun.bsky.social on receiving a F31 award from NIGMS to "Illuminate the Molecular Mechanisms of Replication and Transcription Coordination" during zygotic genome activation and support her training. 0233