Biology subjects

Niekamp, S.

Publications and source records attributed to Niekamp, S..

6 recordsLinked to original sources

bioRxiv2025

Mutual Antagonism Between PRC1 Condensates and SWI/SNF in Chromatin Regulation

Opposing activities of conserved chromatin regulatory complexes, such as the Polycomb Repressive Complex 1 (PRC1) and the activating chromatin remodeler SWI/SNF play critical roles in regulating gene expression during development and differentiation. The mechanisms by which these complexes compete to regulate chromatin states remain poorly understood. We combine single-molecule analysis and genomic approaches in cultured cells to demonstrate that the condensate-forming properties of PRC1 play an important role in excluding SWI/SNF from chromatin. Consistently, PRC1 compositions with a higher propensity for condensate formation are more effective in preventing SWI/SNF binding. Conversely, SWI/SNF-bound chromatin significantly reduces PRC1 binding and subsequent condensate formation. Notably, SWI/SNF can suppress PRC1 condensate formation in an ATP-hydrolysis independent manner. We propose that the condensate properties of different PRC1 compositions drive mutual PRC1-SWI/SNF antagonism to properly balance these competing regulatory activities during development.

biophysics↗

bioRxiv2023

Modularity of PRC1 Composition and Chromatin Interaction define Condensate Properties

Polycomb repressive complexes (PRC) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells and the ability of PRC1 to form condensates has been proposed to contribute to maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. By reconstituting and imaging PRC1 with various subunit compositions, we find that the exact combination of PHC and CBX subunits determine the initiation, morphology, stability, and dynamics of condensates. In particular, the polymerization activity of PHC2 strongly influences condensate dynamics to promote formation of structures with distinct domains that adhere to each other but do not coalesce. Using live cell imaging, we confirmed that CBX properties are critical for condensate initiation and that PHC polymerization is important to maintain stable condensates. Together, we propose that PRC1 can fine-tune the degree and type of condensation by altering its composition which might offer important flexibility of regulatory function during different stages of development.

molecular biology↗

bioRxiv2020

Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule binding domains (MTBD) that step along the track. Here, we simultaneously tracked two MTBDs and one AAA ring of a single dynein, as it undergoes hundreds of steps with nanometer precision using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take different sized steps. This variability in the movement between AAA ring and MTBD results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

biophysics↗

bioRxiv2019

A 6-nm ultra-photostable DNA Fluorocube for fluorescence imaging

Photobleaching limits extended imaging of fluorescent biological samples. Here, we developed DNA origami-based \"Fluorocubes\" that are similar in size to the green fluorescent protein (GFP), have single-point attachment to proteins, have a 50-fold higher photobleaching lifetime and emit 40-fold more photons than single organic dyes. We demonstrate that DNA Fluorocubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.

biophysics↗

bioRxiv2018

Stalk-mediated communication in the dynein motor domain

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~135 [A] coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

biochemistry↗

bioRxiv2017

High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level

To uncover the mechanisms of molecular machines it is useful to probe their structural conformations. Single-molecule Forster resonance energy transfer (smFRET) is a powerful tool for measuring intra-molecular shape changes of single-molecules, but is confined to distances of 2-8 nm. Current super-resolution measurements are error prone at <25 nm. Thus, reliable high-throughput distance information between 8-25 nm is currently difficult to achieve. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with [~]1 nm accuracy at any distance >2 nm, using a standard TIRF microscope and open-source software. We applied our two-color localization method to uncover a [~]4 nm conformational change in the \"stalk\" of the motor protein dynein, revealing unexpected flexibility in this antiparallel coiled-coil domain. These new methods enable high-accuracy distance measurements of single-molecules that can be used over a wide range of length scales.

biophysics↗