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C8. Binding, Intracellular Granules and Droplets - Biology

C8. Binding, Intracellular Granules and Droplets - Biology


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We've studied different types of protein aggregation, including aggregation of the native state (to form dimers, trimers, multimers, filaments) or alternate conformations (such as in prion protein aggregation and formation of inclusion bodies of misfolded proteins). We've seen analogous particles, lipid droplets, which contain TAGs and cholesterol esters surrounded by a phospholipid monolayer with adsorbed protein, also promoted to the state of an organelle (in contrast to the recent demotion of the planet Pluto to a dwarf planet or plutoid). The lipid droplet however are surrounded by a "membrane", in this case a monolayer.

How do these and other granules form. A quick review of the Cell Tutorial (scroll to bottom) shows granule formation can be caused by a classic "phase transition", not unlike gaseous water can self associate through hydrogen bonds to form liquid drops which can freeze with the formation of more hydrogen bonds to form solids. Soluble biomolecules in cells can reversibly aggregate through the summation of multiple weak IMFs to form storage granules. This balance might be perturbed if storage granules aggregate further in a potentially irreversible process with health consequences. We've seen examples of the latter when neurodegenerative diseases like Alzheimer's and Mad Cow Disease. Lets delve into new insights into the processes involved in droplet formation.

section Summer 2017

Imagine small amounts of a sparing soluble oil added to an aqueous solution. Initially it is in solution, but at a higher concentration, London dispersion forces and the “hydrophobic effect” would drive the oil out of solution into liquid drops. This phase separation could also be called liquid-liquid demixing as two liquids (solubilized oil in water and separated oil drops) separate. This process has been shown to produce many types of non-membrane bound droplets (not to be confused with membrane bound vesicles) in the cell.

This phenomenon has also been seen with intrinsically disordered proteins. These are characterized by amorphous structures with repeated, often positively charged amino acids. Under the right condition, these can aggregate and “precipitate” from the solution. What is the nature of the precipitate? It might have properties more like distinct liquid droplets so this process could be called liquid-liquid demixing.

Properties of demixed drops would include reduced rates of diffusion of material into an out of the drop, coupled movements of materials in the drop, and probable weak hydrophobic-dependent aggregation making drops sensitive to agents like detergents. Liquid-like diffusion inside the drop is observed as evident by rapid recovery of fluorescence from partially photobleached internal components of the drop.

As with the formation of a crystalline solid from a liquid solution, the process must be seeded. For intrinsically disordered proteins, this process can be “catalyzed” by poly-(ADP-ribose), a nucleic acid-like polyanion. The negative charges would counter the positive charges in the disordered protein domain, which without neutralization, would interfere with protein/protein contacts necessary for aggregation/droplet formation and demixing. Aggregation in these cases may arise from hydrophobic interactions (even though hydrophobic side chains are underrepresented in the disordered domains).

Solubility of proteins in cells is a fascinating topic in itself. It was recently discovered that the high concentration of ATP (5 mM) in the cell actually helps to solubilize proteins. ATP is considered a hydrotrope. It’s a small molecule with a vary distinct polar part (polyphosphate and ribose) and a more nonpolar part (the adenosine ring). Hence it acts sort of like a mini-detergent (an amphiphle) but it doesn’t form micelles. It does help stabilize more nonpolar parts of proteins in solution and has been shown to inhibit aggregate formation and also disaggregate some aggregates.

Insert: Draw structure of ATP and hydrophobic moment image

Biochemists also use the term gel (examples include polyacrylamide gel or fibrin blood clots which are chemically cross-linked) and a gel in the gel to liquid-crystalline phase transitions in lipid bilayers, held together by weak noncovalent interactions), when they wish to describe a structure that is neither clearly solid nor liquid. A structure like the cytoskeleton or the actin-myosin network would be examples of the latter.

Noncovalent gels would be characterized by regulatable dissociation of subunits and hence short half-lifes. A gel (either covalent or noncovalent) with a high-water content could be called a hydrogel which would contain hydrophilic components. An example would be RNA-protein containing particles

RNA granules

Many of the granules contain RNA and proteins and are called ribonucleoprotein bodies (RNPs) or RNA granules. Specific examples of these include cytoplasmic processing bodies, neuronal and germ granules, as well as nuclear Cajal bodies, nucleoli and nuclear dots/bodies). Some granules just contain proteins , including inclusion bodies with misfolded and aggregated proteins and those with active protein involved in biosynthesis, including the purinosome (for purine biosynthesis) and cellusomes (for cellulose degradation).

Another feature found in some neurodegenerative disease is a trinucleotide repeat. In Fragile X syndrome, there 230-4000 repeats of the CGG codon in the noncoding parts of the genome, compared to less than 50 in the normal gene. In Huntington’s disease, the repeat CAG is found in the protein coding part of the affected gene. The translated protein has a string of glutamines which probably causes protein aggregation. Specific proteins may also bind to the string of CAGs.

If the trinucleotide expansion is in intronic DNA, deleterious effects are not associated with translated proteins but with the transcribed RNA in the nucleus. The intronic repeats would be spliced out of the primary RNA transcript. A CTG DNA repeat would produce a poly CUG containing RNAs (found in myotonic dystrophy), which could aggregate through non-perfect base pairing. In vitro experiment show that small complexes are soluble, but as the size increases, a liquid-liquid demixing phase separation (or alternatively a liquid-gel transition) can occur, forming spherical drop RNA particles. This would explain the observation that pathologies occur above a certain repeat length. If misfolded proteins are also present, these particles might combine to form larger gels.

In the control experiment, when the repeats were scrambled, demixing and spherical particle formation was not observed. In an experiment similar to the addition of 1,6-hexanediol to intrinsically disorderd proteins, if small antisense trinucleotide repeats, such as (CTG)8, which could interfere with the weak H bonds between G and C in the aggregates, were added, the size of RNA drops (foci) were reduced. In vivo experiments showed characteristic drop-like structures but only if the repeats were of sufficient size.

Researchers found that in vitro, RNA drop formation was inhibited by monovalent cations. In the presence of 0.1 M ammonium acetate, which permeates cells without affecting pH, 47× CAG RNA droplets in vitro disappeared.

Aggregation of mRNA might be one way to regulate its translation and hence indirectly regulate gene activity. There are advantages to regulating the translation of a protein from mRNA, especially if the "activity" of the mRNA could be dynamically regulated. This would be useful if new protein synthesis was immediately required. Hence one way to regulate mRNA activity (other than degradation) is through reversible aggregation.

Protein drops and granules

The cytoskeletal proteins actin and tubulin (heterodimer of alpha and beta chains) can exist in soluble (by analogy to water gaseous) states or in condensed filamentous state (actin filaments and microtubules respectively). GTP hydrolysis is required for tubulin formation. Actin binds ATP which is necessary for filament formation but ATP cleavage is required for depolymerization. Hence nucleotide binding/hydrolysis regulates the filament equilibrium which differentiates from simple phases changes such as in water.

Since only certain proteins form granules, they must have similar structural features that facilitate reversible binding interactions. There appear to be multiple sites with these protein that individually form weak binding interactions, but collectively through multivalent (multiple) binding interactions allow robust but not irreversible granule formation. Here are some characteristics of proteins found in granules:

  • the protein NCK has 3 repeated domains (SH3) the bind to proline-rich motifs (PRMs) in the protein NWASP. These proteins are involved in actin polymerizaiton. In high concentration they precipitate from solution and coalesce to form larger droplets;
  • repeating interaction domains are widely found especially among RNA binding proteins;
  • some proteins contain Phe-Gly (FG) repeats separated by hydrophilic amino acids in portions of the protein that are intrinsically disordered.
  • a biotinylated derivative of 5-aryl-isoxazole-3-carboxyamide (structure below) precipitates proteins which are enriched in those that bind RNA (RBPs). In general the precipitate proteins were intrinsically disordered characterized by low complexity sequences (LCS). One such example contained 27 repeats of the tripeptide sequence (G/S)Y(G/S). The proteins could also form hydrogels (made of hydrophilic polymers and crosslinks) and transition between soluble and gel phases with extensive hydrogen bond networks. The hydrogel gel phase gave x-ray diffraction patterns similar to beta structure-enriched amyloid proteins. Short ranged weak interactions between LCS might then drive reversible condensation to gel like granule states characterized by extensive hydrogen bonding (again similar to hydrogen bonding on ice formation). If this process goes awry, more continued and irreversible formation of a solid fibril (as seen in neurodegenerative diseases) might occur from the hydrogel state;

  • RNAs appear in granules as protein bind them through RNA binding domains of proteins which interact through low complexity sequences leading to phase separation and hydrogel-like formation of granules. Around 500 RNA binding proteins have been found in the human RNA interactome. They are enriched in LCSs and have more tryosines than average proteins in the whole proteome in which the Tyr are often found in an (G/S)Y(G/S) motif. Phosphorylation of tyrosines (Y) in LCS may decrease association and hydrogel stability.

Given the many neurodegenerative diseases are associated with unfolded/misfolded protein aggregates, the high protein concentrations in protein-containing liquid drops might pose problems to cells. If high enough, the equilibrium might progress from the liquid drop to a solid precipitate, which would have severe cellular consequences. The progression to the solid state may irreversibly affect the cell.

In the next section, 5D: Binding and the Control of Gene Transcription, we will explore how liquid-liquid demixing can help explain chromatin structure and dynamics.


The embryo of any organism that reproduces sexually must develop germ cells, such as those that go on to become egg and sperm cells in animals. This is because these are the only cells that are destined to transfer genetic material to the next generation. One characteristic of developing germ cells is the presence of particles termed “germ granules” (Voronina et al., 2011). Made from various RNA and protein molecules, these granules are believed to regulate the translation of messenger RNA (mRNA) molecules inside the germ cells during development (Seydoux and Braun, 2006).

Many of the components that are found in germ granules are conserved between distantly related species. Studies in this area have commonly involved the roundworm Caenorhabditis elegans, which, like other animals, starts life as a single fertilized egg or zygote. At first, germ granules are spread uniformly throughout this cell. However, as the zygote starts to develop a distinct front and back, the germ granules are only found in the back of the zygote: this is why the germ granules in C. elegans are called P granules (with “P” being short for the P lineage of cells that forms at the posterior). This process is repeated during further cell divisions, such that the P granules continue to segregate into those cells that will eventually give rise to the germ cells. Now, in eLife, Geraldine Seydoux and colleagues at the Johns Hopkins University School of Medicine – including Jarrett Smith as first author – report how two RNA-binding proteins with opposing effects control where P granules form (Smith et al., 2016).

Early explanations as to why P granules segregated asymmetrically were based on the idea that they were actively transported to the posterior half. However, a few years ago, it was noted that proteins found in germ granules could spontaneously de-mix from the cytoplasm and coalesce to form germ granules (Brangwynne et al., 2009). This phenomenon, called a phase transition, resembles how oil droplets form when oil is mixed with water. However, only the granules that formed in the posterior of the zygote were stable in C. elegans, and any granules that started to form in the front half disappeared instead.

P granules only grow in the posterior, in part, because a gradient of RNA-binding proteins somehow restricts where they can form (Griffin et al., 2011 Schubert et al., 2000). This raises some questions: how is a protein gradient transformed into an on-off switch for P granule formation? And what triggers the phase transition so that P granules are only stable in the posterior?

Some proteins in germ granules contain “intrinsically disordered regions” that lack a well-defined three-dimensional structure (Kato et al., 2012 Courchaine et al., 2016 Hyman et al., 2014). Smith et al. now demonstrate that two intrinsically disordered, RNA-binding proteins – namely MEG-3 and its homolog MEG-4 – lie at the heart of P granule formation, and that MEG-3 is essential for germ granules to nucleate. In vitro, MEG-3 will spontaneously assemble into aggregates, but only at concentrations higher than those found in the zygote (Figure 1). However, Smith et al. discovered that this phase transition was enhanced when RNA is present. As such, simply varying the RNA levels in a test tube or in the zygote can change when and where P granules form. Smith et al. also showed that another RNA-binding protein called MEX-5 (which is not a component of P granules) competes with MEG-3 for access to the RNA, and that the high concentrations of MEX-5 at the front end of the zygote prevent P granules being formed there (Figure 1).

RNA and the formation of P granules.

(A) The formation of liquid droplets of the protein MEG-3 (red circles) in vitro is enhanced by RNA (second and fourth panels) and antagonized by the protein MEX-5 (third panel). (B) In the single-celled zygote, the front of the cell (left) has higher levels of MEX-5 (blue shading) than the rear of the cell (right). MEX-5 and MEG-3 both bind to RNA, and competition between them restricts the formation of P granules to the regions where the concentration of MEX-5 is low (that is, to the posterior end of the cell). (C) If the RNA levels in the cell (represented by the area of the gray bar) are increased (by blocking an RNA degradation pathway), more P granules are formed, and they also form further forward in the zygote than normal.

FIGURE CREDIT: Alexey Soshnev, Tatjana Trcek and Ruth Lehmann.

A recent theoretical study in C. elegans proposed a similar mechanism, with MEX-5 and a P granule protein called PGL-3 competing to bind to mRNA molecules (Saha et al., 2016). However, Smith et al. show that PGL-3 is not essential for the nucleation of P granules, and that it is not needed to establish the asymmetric distribution of granules either. It is therefore more likely that MEG-3 forms a critical scaffold for the P granule and then recruits other P granule proteins, including PGL-3 (Hanazawa et al., 2011 Wang et al., 2014).

MEX-5 and MEG-3 bind to RNA with little specificity (Pagano et al., 2007 Smith et al., 2016), but the adaptor proteins found in germ cells might make it possible for these proteins to bind to different sets of mRNAs (Weidmann et al., 2016). This selective binding could establish a gradient of specific mRNAs that runs from the front to the back of the zygote, with critical mRNAs being captured at the end of the cell that goes on to become the germ cells (Gallo et al., 2010 Lehmann, 2016 Seydoux and Braun, 2006).

RNA-protein granules are widespread in nature. They are, in fact, found in every cell in the human body, and likely regulate RNAs in many different ways (Couchrane, et al., 2016). Phase transitions might drive the formation of these other granules too, similar to P granule formation in C. elegans. These granules often contain RNA-binding proteins with intrinsically disordered regions and are also enriched with RNAs (Han et al., 2012 Lin et al., 2015 Schwartz et al., 2013 Teixeira et al., 2005 Zhang et al., 2015). As such, many of them may likewise rely on RNAs to form. The new mechanism reported by Smith et al. could explain how a variety of RNA-protein granules end up sorted into different areas of the cell, even though they share multiple components.


RNA Droplets

Liquid–liquid phase separation is emerging as the universal mechanism by which membraneless cellular granules form. Despite many previous studies on condensation of intrinsically disordered proteins and low complexity domains, we lack understanding about the role of RNA, which is the essential component of all ribonucleoprotein (RNP) granules. RNA, as an anionic polymer, is inherently an excellent platform for achieving multivalency and can accommodate many RNA binding proteins. Recent findings have highlighted the diverse function of RNA in tuning phase-separation propensity up or down, altering viscoelastic properties and thereby driving immiscibility between different condensates. In addition to contributing to the biophysical properties of droplets, RNA is a functionally critical constituent that defines the identity of cellular condensates and controls the temporal and spatial distribution of specific RNP granules. In this review, we summarize what we have learned so far about such roles of RNA in the context of in vitro and in vivo studies.


Acknowledgements

We thank J. Onuchic and S. Padrick for discussion of the theoretical aspects of this study, L. Rice for sharing his fluorescence microscope, M. Socolich for a gift of purified eGFP, K. Luby-Phelps and A. Bugde for advice on FRAP experiments, S. Padrick and L. Doolittle for help in purifying actin and the Arp2/3 complex and for sharing reagents, N. Grishin and S. Shi for help with database searches, K. Lynch for providing the PTB expression construct, D. Billadeau and T. Gomez for providing antibodies, A. Ramesh, W. Winkler and P.-L. Tsai for advice on RNA experiments, K. Roybal and C. Wülfing for sharing unpublished data, and J. Liu for help with cryo-electron tomography. This work was supported by the following: the Howard Hughes Medical Institute and grants from the National Institutes of Health (NIH) (R01-GM56322) and Welch Foundation (I–1544) to M.K.R., a Chilton Foundation Fellowship to H.-C.C., an NIH EUREKA award (R01-GM088745) to Q.-X.J., an NIH Cancer Biology T32 Training Grant to M.L., a National Science Foundation award (DMR-1005707) to P.S.R. and a Gates Millennium Fund award to J.V.H. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract number W-31-109-ENG-38. BioCAT is NIH-supported Research Center RR-08630.


3 VIRUSES

Hepatitis C virus (HCV) is one of the most widely studied infectious agents in terms of interactions with LDs. The life cycle of this virus is closely tied to lipid metabolism, as viral particles circulating in the blood of infected patients are bound to lipoproteins, forming lipoviroparticles (Boyer et al., 2014 ). In HCV-infected cells, the nucleocapsid (core) protein and a nonstructural protein of the viral replication complex, NS5A, are localized to LDs (Miyanari et al., 2007 ). The visualization of virus assembly in HCV-infected cells has proved challenging, but, by overproducing the structural proteins (the core and the two envelope proteins), virus-like particles can be the observed budding at the ER membranes in close association with LDs (Hourioux et al., 2007a Roingeard, Hourioux, Blanchard, & Prensier, 2008 Figure 1a). It is currently thought that HCV formation involves the use of LDs as assembly platforms for the virus, with the core protein playing a key role in this mechanism. The core protein is the first protein translated from the viral RNA and is released from the single viral polyprotein encoded by the viral genome, through two consecutive cleavage events: the first, mediated by the signal peptidase and the second by signal peptide peptidase. These events generate a mature protein that diffuses laterally in the ER membranes towards the surface of the LDs (McLauchlan, Lemberg, Hope, & Martoglio, 2002 ). The HCV core protein interacts physically with DGAT1 in the ER membrane, and this interaction, coupled to active DGAT1 triglyceride synthesis, is required for the localization of core to LDs (Herker et al., 2010 ). Unlike core, which is found almost entirely on LDs, NS5A is found on both LDs and ER membranes. DGAT1 also interacts with NS5A, probably functioning as a molecular bridge between core and NS5A to ensure their targeting to the same LD (Camus et al., 2013 ). Two other cell factors, tail-interacting protein 47 and the Ras-related protein Rab18, which are associated with LDs in hepatocytes, interact with NS5A and contribute to HCV formation (Vogt et al., 2013 Salloum, Wang, Ferguson, Parton, & Tai, 2013 ). Rab18 may promote the physical association of NS5A with other components of the viral replication machinery and LDs (Salloum et al., 2013 ). HCV assembly probably involves the close apposition of LDs against viral replication sites located in specialized regions of ER membranes and generated by the nonstructural proteins (Ferraris et al., 2013 ). NS5A, which has RNA-binding properties, may transport viral RNA from the replication sites to LDs for interaction with core, leading to the encapsidation of the newly synthesized viral RNA and the formation of virions. Very low-density lipoproteins (VLDL) are assembled in the luminal compartment of the ER, and most of the lipids used for their production are derived from LDs. The nascent HCV particles, therefore, probably follow the VLDL assembly pathway, to generate virions with incorporated apolipoproteins. HCV/LD interaction is not restricted to viral morphogenesis, as chronic HCV infection is linked to LD accumulation, or steatosis, in the liver of patients with chronic HCV infection (Roingeard & Hourioux, 2008 ). This steatosis can affect the natural course of the infection, aggravating the progression of hepatic fibrosis. Levels of LD accumulation in HCV-infected cells have been shown in vitro to be directly linked to polymorphisms of the core protein sequence (Hourioux et al., 2007b ), although host genetic factors are the principal factors controlling the severity of liver steatosis in vivo (Roingeard, 2013 ).

GB virus B, which is closely related to HCV and causes acute hepatitis in experimentally infected tamarins, encodes a core protein that colocalizes with LDs, due to a region similar to the HCV core protein (Hourioux et al., 2007a ). Interaction with LDs is not unique to HCV and related viruses from the Flaviviridae family, as the core protein of dengue virus (DENV) has also been shown to localize with LDs, although these viruses infect different host cells (hepatocytes for HCV, mosquito and human monocytes, and macrophages for DENV Samsa et al., 2009 ). A nonstructural protein from DENV, NS3, cooperates with Rab18 to recruit the host fatty acid synthetase to sites of viral replication (Heaton et al., 2010 Tang, Lin, Liao, & Lin, 2014 ). Interestingly, the DENV core protein has recently been shown to interact specifically with VLDL, suggesting that DENV may also form lipoviroparticles (Faustino et al., 2014 ). Although much work remains to be done in the DENV model, core protein/LD association appears to be crucial for virus production in both cases.

Rotaviruses replicate in enterocytes and have been shown to highjack LDs for their own purposes (Figure 1b). Early stages of viral assembly and replication take place in virus-induced cytoplasmic inclusion bodies called viroplasms, from which double-layered particles (DLPs) are released. These particles acquire an outer layer from the rough ER, to form triple-layered particles (Trask, McDonald, & Patton, 2012 ). These triple-layered particles contain four major capsid proteins (VP2, VP4, VP6, and VP7) and two minor proteins (VP1 and VP3). Mature virions, which are non-enveloped viruses, are thought to be released through the exocytosis pathway, after removal of the ER membrane. Release from the infected cell exposes the virion to gastrointestinal tract proteases, resulting in the cleavage of VP4 into VP5 and VP8, for which the production of fully infectious virions (Trask et al., 2012 ). The viroplasm, which contains an active RNA replication complex and is essentially formed by two nonstructural proteins NSP2 and NSP5, colocalizes with LDs in infected cells (Cheung et al., 2010 ). LDs recruitment close to the viroplasm begins soon after initial infection, and the number of viroplasm-LD complexes increases during the replication cycle (Cheung et al., 2010 ). Lipidome analysis has shown that the total lipid content of the cell increases during rotavirus infection, consistent with an increase in the interaction of LDs with viroplasms (Gaunt et al., 2013a ). Chemical compounds blocking fatty-acid synthesis or interfering with LD homeostasis, such as triacsin C, have been shown to decrease the number and size of viroplasms and the number of infectious viruses produced (Gaunt, Cheung, Richards, Lever, & Desselberger, 2013b ).

Finally, different viral proteins from various viral models, including the μ1 outer capsid protein of reoviruses (Coffey et al., 2006 ) and the agnoprotein of the polyomavirus BK (Unterstab et al., 2010 ), have been shown to interact with LDs. The biological relevance of these associations remains to be determined in these viral models, but these findings suggest that various viruses have evolved mechanisms for interacting with LDs and, possibly, for subverting the function of these organelles for their use as a platform for viral particles assembly.


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Department of Pharmacology, Boston University School of Medicine, Boston, MA, USA

Department of Neurology, Boston University School of Medicine, Boston, MA, USA

Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA, USA

Department of Medicine, Harvard Medical School, Boston, MA, USA

The Broad Institute of Harvard and MIT, Cambridge, MA, USA

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B.W. researched data for the article. B.W. and P.I. provided substantial contributions to discussion of the article’s content, wrote the article, and reviewed and edited the manuscript before submission.

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RNA Droplets

Liquid–liquid phase separation is emerging as the universal mechanism by which membraneless cellular granules form. Despite many previous studies on condensation of intrinsically disordered proteins and low complexity domains, we lack understanding about the role of RNA, which is the essential component of all ribonucleoprotein (RNP) granules. RNA, as an anionic polymer, is inherently an excellent platform for achieving multivalency and can accommodate many RNA binding proteins. Recent findings have highlighted the diverse function of RNA in tuning phase-separation propensity up or down, altering viscoelastic properties and thereby driving immiscibility between different condensates. In addition to contributing to the biophysical properties of droplets, RNA is a functionally critical constituent that defines the identity of cellular condensates and controls the temporal and spatial distribution of specific RNP granules. In this review, we summarize what we have learned so far about such roles of RNA in the context of in vitro and in vivo studies.


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Present address: Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, 94702, USA

Hideki Nakamura and Albert A. Lee: These authors contributed equally to this work.

Affiliations

Department of Cell Biology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 21205, USA

Hideki Nakamura, Albert A. Lee, Shigeki Watanabe, Shiva Razavi, Allister Suarez, Yu-Chun Lin, Makoto Tanigawa, Robert DeRose, Diana Bobb & Takanari Inoue

Center for Cell Dynamics, Institute for Basic Biomedical Sciences, Johns Hopkins University, Baltimore, Maryland, 21205, USA

Hideki Nakamura, Albert A. Lee, Elmer Rho, Allister Suarez, Yu-Chun Lin, Brian Huang, Robert DeRose, Diana Bobb & Takanari Inoue

Center for Imaging Science, Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, Maryland, 21218, USA

Ali Sobhi Afshar & John Goutsias

Department of Biomedical Engineering, Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, Maryland, 21218, USA

Shiva Razavi, Makoto Tanigawa & Takanari Inoue

Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 21205, USA

William Hong & Sandra B. Gabelli

Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 21287, USA

Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 21287, USA