Publications by authors named "Jorine M Eeftens"

8 Publications

  • Page 1 of 1

Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Cell 2020 04;181(2):306-324.e28

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA; Howard Hughes Medical Institute, Princeton, NJ 08544, USA. Electronic address:

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
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http://dx.doi.org/10.1016/j.cell.2020.03.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816278PMC
April 2020

Distinct Roles for Condensin's Two ATPase Sites in Chromosome Condensation.

Mol Cell 2019 12 16;76(5):724-737.e5. Epub 2019 Oct 16.

Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address:

Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.
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http://dx.doi.org/10.1016/j.molcel.2019.09.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900782PMC
December 2019

Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism.

EMBO J 2017 12 8;36(23):3448-3457. Epub 2017 Nov 8.

Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, The Netherlands

Condensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction can proceed in large steps, driving DNA molecules into a fully condensed state against forces of up to 2 pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt insensitive and for the subsequent compaction process. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.
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http://dx.doi.org/10.15252/embj.201797596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709735PMC
December 2017

The condensin complex is a mechanochemical motor that translocates along DNA.

Science 2017 11 7;358(6363):672-676. Epub 2017 Sep 7.

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. We used single-molecule imaging to demonstrate that condensin is a molecular motor capable of adenosine triphosphate hydrolysis-dependent translocation along double-stranded DNA. Condensin's translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.
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http://dx.doi.org/10.1126/science.aan6516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862036PMC
November 2017

Condensin Smc2-Smc4 Dimers Are Flexible and Dynamic.

Cell Rep 2016 Mar 18;14(8):1813-8. Epub 2016 Feb 18.

Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft 2628 CJ, the Netherlands. Electronic address:

Structural maintenance of chromosomes (SMC) protein complexes, including cohesin and condensin, play key roles in the regulation of higher-order chromosome organization. Even though SMC proteins are thought to mechanistically determine the function of the complexes, their native conformations and dynamics have remained unclear. Here, we probe the topology of Smc2-Smc4 dimers of the S. cerevisiae condensin complex with high-speed atomic force microscopy (AFM) in liquid. We show that the Smc2-Smc4 coiled coils are highly flexible polymers with a persistence length of only ∼ 4 nm. Moreover, we demonstrate that the SMC dimers can adopt various architectures that interconvert dynamically over time, and we find that the SMC head domains engage not only with each other, but also with the hinge domain situated at the other end of the ∼ 45-nm-long coiled coil. Our findings reveal structural properties that provide insights into the molecular mechanics of condensin complexes.
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http://dx.doi.org/10.1016/j.celrep.2016.01.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785793PMC
March 2016

Copper-free click chemistry for attachment of biomolecules in magnetic tweezers.

BMC Biophys 2015 25;8. Epub 2015 Sep 25.

Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft, Delft, The Netherlands.

Background: Single-molecule techniques have proven to be an excellent approach for quantitatively studying DNA-protein interactions at the single-molecule level. In magnetic tweezers, a force is applied to a biopolymer that is anchored between a glass surface and a magnetic bead. Whereas the relevant force regime for many biological processes is above 20pN, problems arise at these higher forces, since the molecule of interest can detach from the attachment points at the surface or the bead. Whereas many recipes for attachment of biopolymers have been developed, most methods do not suffice, as the molecules break at high force, or the attachment chemistry leads to nonspecific cross reactions with proteins.

Results: Here, we demonstrate a novel attachment method using copper-free click chemistry, where a DBCO-tagged DNA molecule is bound to an azide-functionalized surface. We use this new technique to covalently attach DNA to a flow cell surface. We show that this technique results in covalently linked tethers that are torsionally constrained and withstand very high forces (>100pN) in magnetic tweezers.

Conclusions: This novel anchoring strategy using copper-free click chemistry allows to specifically and covalently link biomolecules, and conduct high-force single-molecule experiments. Excitingly, this advance opens up the possibility for single-molecule experiments on DNA-protein complexes and molecules that are taken directly from cell lysate.
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http://dx.doi.org/10.1186/s13628-015-0023-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582843PMC
September 2015

CSF levels of DJ-1 and tau distinguish MSA patients from PD patients and controls.

Parkinsonism Relat Disord 2014 Jan 12;20(1):112-5. Epub 2013 Sep 12.

Department of Neurology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands; Department of Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands. Electronic address:

Differential diagnosis between Parkinson's disease (PD) and multiple system atrophy (MSA) is difficult, particularly at early disease stages, but is important for therapeutic management. The protein DJ-1 is implicated in the pathology of PD but little is known about its involvement in MSA. We aimed to determine the diagnostic value of CSF DJ-1 and tau proteins for discriminating PD and MSA. DJ-1 and total tau levels were quantified in the CSF of 43 PD patients, 23 MSA patients and 30 non-neurological controls matched for age and gender. Patients were part of a study with a 3-year prospective design with extended case-review follow-up of up to 9 years, ensuring maximum accuracy of the clinical diagnosis. Our results showed that CSF DJ-1 levels could distinguish MSA from PD with a 78% sensitivity and 78% specificity (AUC = 0.84). The combination of DJ-1 and tau proteins significantly improved this discrimination to 82% sensitivity and 81% specificity to identify MSA from PD (AUC = 0.92). Our results highlight the potential benefits of a combination of DJ-1 and total tau as biomarkers for differential diagnosis of MSA and PD.
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http://dx.doi.org/10.1016/j.parkreldis.2013.09.003DOI Listing
January 2014

Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome.

Hum Mol Genet 2013 Mar 21;22(5):852-66. Epub 2012 Nov 21.

Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Nijmegen, the Netherlands.

Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.
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http://dx.doi.org/10.1093/hmg/dds490DOI Listing
March 2013