ABSTRACT

This report summarizes an international conference on molecular machines convened at New York University, Abu Dhabi by Piergiorgio Percipalle, George Shubeita and Serdal Kirmizialtin. The meeting was conceived around the epistemological question of what do we understand, or not understand (if we have open minds), about the degree to which cells operate by the individual actions of single enzymes or non-catalytic protein effectors, versus combinations of these in which their heterotypic association creates an entity that is more finely tuned and efficient – a machine. This theme was explored through a vivid series of talks, summarizing the latest findings on macromolecular complexes that operate in the nucleus or cytoplasm.

Introduction

In 2010, New York University in the U.S. took the bold step to establish a full liberal arts campus with a strong research focus in Abu Dhabi, in the United Arab Emirates (NYUAD). As part of its continuing momentum, in April, 2019 it convened an international meeting on ‘Nuclear and cytoplasmic molecular machines at work’, organized by NYUAD faculty members Piergiorgio Percipalle, George Shubeita and Serdal Kirmizialtin.

Conference poster drawn by George Shubeita, New York University, Abu Dhabi.

Conference poster drawn by George Shubeita, New York University, Abu Dhabi.

The late 19th and early 20th century notion, in the field then called physiological chemistry, that cells typically run on solitary enzymes was in due course supplanted by the discovery that heterotypic protein complexes are far more often the agents. The science of deciphering these cellular machines has been a profound epistemological change in modern cell biology, and this meeting brought together researchers tackling this problem by using a range of experimental and theoretical approaches.

Before describing the meeting, some broader dimensions warrant mention. Hardly any of the participants had been in the United Arab Emirates before. We were struck by the international mix of students we encountered and how vibrant and outgoing they were. In an engaging opening welcome, the provost, Fabio Piano, informed us that NYUAD has a student acceptance rate of less than 5% and that ∼85% of the student population comes from outside the United Arab Emirates, notably from more than 100 different countries.

Scientific program

The first session focused on the functions and dynamics of nuclear actin. Robert Grosse (University of Freiburg, Germany) discussed how specific signaling inputs can trigger different types of nuclear F-actin formation. He showed that Ca2+ transients rapidly stimulate linear actin polymerization in a process that induces changes in the chromatin. He also discussed the involvement of nuclear F-actin in mitotic spindle formation, as well as post-mitotic chromosome decompaction and nuclear volume expansion (Wang et al., 2019a).

Maria Vartiainen (University of Helsinki, Finland) described the use of two complementary mass spectrometry approaches they used to identify different types of binding partners for nuclear actin (Viita et al., 2019). She showed that actin interacts with all transcribed genes, as well as identified novel actin-binding partners and discussed the multifunctional nature of nuclear actin. Piergiorgio Percipalle (NYU, Abu Dhabi, United Arab Emirates) presented his work on the role of actin in chromatin regulation. He demonstrated that nuclear actin controls the deposition of the ATPase Brg1, a component of the chromatin remodeling complex SWI/SNF (BAF), in a process that contributes to the regulation of gene programs and the establishment of cellular identity (Xie et al., 2018). He also presented new findings on the role of nuclear β-actin in cell differentiation.

Irene Chiolo (University of Southern California, Los Angeles, USA) described a role for nuclear actin filaments and myosins in transporting sites of homologous recombination repair of double-strand DNA breaks (DSBs) to the nuclear periphery (Caridi et al., 2018). This pathway involves Arp2/3 nucleating filamentous actin at the DSBs, and defects in this pathway impair DSB repair.

Roland Foisner (Medical University of Vienna, Austria) presented results on the intranuclear pool of lamins, including the key finding that they interact with active chromatin. He provided additional evidence that these nucleoplasmic lamins influence the mobility of euchromatic regions of the genome and their accessibility to regulatory factors.

The advent of new technology to track genomic loci was the topic of the talk by Tom Misteli (National Cancer Institute, Bethesda, USA) who presented a ‘deep dive’ into single-cell interrogation of 3D chromatin–chromatin interactions (Finn et al., 2019). The surprising finding was that previous ensemble studies have vastly under-estimated the population prevalence of these interactions. Thoru Pederson (University of Massachusetts Medical School, Worcester, USA) described the latest in a series of CRISPR-based genome labeling platforms his group has developed, termed CRISPR Sirius, and showed how it has been recently deployed to resolve unanticipated cell cycle changes in the intranuclear mobility of an interrogated genomic locus (Ma et al., 2019).

The specificity of CRISPR-based gene editing, the major challenge in the application of this technology in the therapeutic domain, was discussed by Rick Russell (University of Texas at Austin, USA). Focusing on the Cas12a nuclease, he presented a kinetic analysis that revealed a two-step enzymatic process, with DNA cleavage being orders of magnitude faster than dissociation of the complex from the target (Strohkendl et al., 2018). Further analysis revealed that R-loop propagation is highly reversible until it is almost complete. This work thus provides a mechanistic basis for the highly specific recognition of the target site by Cas12a.

Ioan Andricioaei (University of California, Irvine, USA) presented molecular dynamics simulations of nucleic acid conformational transitions during their passage through protein-based pores (Xu et al., 2018). These included local changes between Watson–Crick versus Hoogsteen conformations, as well as global transitions, such as topoisomerase-based DNA supercoiling relaxation, dendrimeric DNA condensation and DNA minicircle formation upon phage ejection.

DNA compaction at the chromosomal level, from bacteria to humans, is mediated by the condensins, but, as discussed by Sevinc Ercan (New York University, New York, USA), in Caenorhabditis elegans, the condensin variant SMC-4 takes on an additional role in X chromosome transcriptional dosage compensation. By deletion and repositioning of the DNA recruitment sites for this machine, she showed that multiple sites are required for transcriptional repression and that the condensin complex moves bidirectionally across megabase-sized DNA domains, representing some of the longest distances traversed by a DNA-based motor.

The issue of kinetic competition between RNA polymerase II elongation and pre-mRNA splicing was addressed by Maria Carmo-Fonseca (University of Lisbon, Portugal). She showed live-cell measurements of intron lifetimes in single pre-mRNAs, which revealed that splicing can take place only seconds after the splice sites are transcribed. Their additional results indicated that the degree to which transcription and splicing are temporally coordinated is based on formation of relatively stable complexes between RNA polymerase II and the spliceosome.

Neus Visa (The Wenner Gren Institute, Stockholm University, Sweden) spoke about her recent study demonstrating that transcription takes place at DNA double-strand breaks, and that their repair requires clearance of RNAs from the damaged locus (Domingo-Prim et al., 2019). She provided evidence that EXOSC10, the catalytic subunit of the RNA exosome, is a key player in this process, which is a prerequisite for assembly of the repair machinery.

The discussion of biophysical approaches to study nuclear motors provided additional depth to the meeting. Michael Feig (Michigan State University, East Lansing, USA) offered physicochemical perspectives on how crowded cellular environments may affect protein structure stability and diffusion by using large-scale molecular simulations of a model of a bacterial cytoplasm. He also discussed how RNA polymerase traffics across DNA. Serdal Kirmizialtin (NYU, Abu Dhabi, United Arab Emirates) presented advances achieved by computational analysis on how metal ions influence the fidelity of a retroviral machine, the HIV reverse transcriptase.

Marina Lusic (University of Heidelberg, Germany) discussed the process by which HIV proviral DNA integrates preferentially at a subset of actively expressed genes in CD4 T-cells that lie adjacent to super enhancers (Lucic et al., 2019). She further reported that these particular genes become clustered into a specific intranuclear compartment upon T-cell activation, with this clustering and high transcriptional activity being major factors in specifying sites of HIV integration.

The presentations on molecular machines that operate in the cytoplasm were equally thought provoking. Paolo de Los Rios (École Polytechnique de Lausanne, Switzerland) described the remarkable finding that chaperones can use the energy derived from ATP hydrolysis to stabilize the native fold of their substrates, even in conditions when this state is thermodynamically unfavorable (Goloubinoff et al., 2018). The work of Paul Whitford (Northeastern University, Boston, USA) on the ribosome provided a striking example of how the combination of modeling strategies and high-resolution structural data can elucidate the dynamics of macromolecular machines.

Many of the other talks focused on the cytoskeletal motors that traffic cargoes in the cytoplasm. Ahmet Yildiz (University of California, Berkeley, USA) described combining single-molecule fluorescence imaging and cryo-electron microscopy to reveal how the human dynein motor moves towards the minus ends of microtubules (Can et al., 2019). Dynein was also the subject of the talk by Simon Bullock (MRC Laboratory of Molecular Biology, Cambridge, UK), in which he described how dynein is switched on by a stem-loop structure in one of its mRNA cargoes (McClintock et al., 2018). He also presented their ongoing efforts to understand how the motor can localize mRNA species to different sites in the same cell. Steven Gross (University of California, Irvine, USA) showed that dyneins engaged in lipid droplet transport can dynamically upregulate their force production in response to impediments in the cytoplasm and that this process involves the NudE and Lis1 proteins. George Shubeita (NYU, Abu Dhabi, United Arab Emirates) presented the unexpected finding that the movement of ‘teams’ of kinesin motors towards microtubule plus-ends is modulated by macromolecular crowding in the cytoplasm, whereas transport by individual motors is not. Further studies of this emergent behavior are likely to shed light on how multiple motors co-operate during transport. Jeremy Teo (NYU, Abu Dhabi, United Arab Emirates) focused on a key output of cytoskeletal motors – regulation of a cell's structural integrity – and showed how this process can be studied using quantitative analysis of time-lapse image series.

Jungseog Kang (New York University, Shanghai, China) presented an analysis of the spindle assembly checkpoint, which provides a quality control step for chromosome–microtubule attachment errors during mitosis and facilitates chromosome alignment (Yang et al., 2019). He described how the spindle assembly checkpoint kinase Mps1 facilitates chromosome alignment by phosphorylating microspherule protein 1 (MCRS1), which promotes efficient recruitment of the kinesin KIF2A to the minus ends of spindle microtubules, thus tuning their length.

The talks on the experimental analysis of motor mechanism and function were complemented by a talk discussing theoretical approaches. Dave Thirumulai (University of Texas at Austin, USA) presented a theory that lays out the structural and chemical basis of the stepping dynamics of myosin V on actin filaments (Hathcock et al., 2020). Changbong Hyeon (Korea Institute for Advanced Study, Seoul, South Korea) discussed new thermodynamic principles quantifying transport efficiency and showed how evolution has shaped cytoskeletal motors to operate with high speed and leverage their energy resources in a highly effective manner (Hwang and Hyeon, 2018).

Steven Block (Stanford University, USA) delivered two lucid and lively talks at the meeting. The first was a well-attended lecture for NYUAD faculty and other guests on the development and application of optical tweezers. Lectures aimed at a general audience require exceptional organizational and oratorical skill, and he succeeded in both respects. At the meeting itself, he spoke on the use of this technology to directly visualize the structural dynamics of single RNA molecules in real time. To that end, a number of tour-de-force experiments were presented, including the use of simultaneous optical trapping and FRET measurements to map the folding-energy landscape of single riboswitches and the relationship to function.

Finally, two talks highlighted how insights gained from fundamental studies of molecular machines might be translated into applied or clinical settings. Zhisong Wang (National University of Singapore, Singapore) described prototypical synthetic DNA-based motors for nanotechnology-based applications (Wang et al., 2019b), while Mauro Giacca (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy and King's College London, UK) demonstrated that the delivery of microRNAs that stimulate proliferation of cardiomyocytes could induce cardiac regeneration after myocardial infarction in mice and large animals (Gabisonia et al., 2019). However, he also emphasized that such small-RNA-based therapy must be tightly controlled to avoid undesired side effects in the long term.

Concluding remarks

The meeting left the participants with a keen sense of how these machines have been refined during evolution, with a precise tuning of their inherent enzymatic properties and the allosteric regulation among each machine's components. In addition, a fascinating issue that arose from the meeting discussions was that the different activities within a molecular machine are in some cases coordinated, as for example might be the case when nuclear actin and RNA polymerases collaborate. Perhaps this meeting has ushered in a new era in which the eukaryotic cell is increasingly viewed in unitary terms, and where researchers feel an increased momentum and sense of an open, sharing community. Several issues were on the minds of everyone as the meeting adjourned. Foremost among these are how the dynamic activities of these fascinating machines be visualized in their native environment in living cells at high spatial and temporal resolution. A longer term objective will be to apply these conceptual and technological approaches to investigate machine dynamics within tissues and organs in animal models of disease.

Acknowledgements

The meeting was sponsored by the NYU Abu Dhabi Research Institute and partly by the International Center for Genetic Engineering and Biotechnology, Trieste.

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Competing interests

The authors declare no competing or financial interests.