RNA and the RNA-binding protein FUS act in concert to prevent TDP-43 spatial segregation.

FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.

Descemet Membrane Endothelial Keratoplasty after failed penetrating keratoplasty- case series and review of the literature.

BACKGROUND: This study aims to evaluate visual outcome, central corneal thickness, and re-bubbling rate in a cohort with undersized sequential Descemet Membrane Endothelial Keratoplasty (DMEK) due to endothelial graft decompensation following primary penetrating keratoplasty (PK). METHODS: All patients who received a sequential DMEK (n = 16) or triple DMEK (n = 2) after failed primary PK between November 2020 and June 2022 were retrospectively evaluated. Analyzed parameters were corrected distance visual acuity (CDVA), central corneal thickness (CCT), re-bubbling rate and graft survival. RESULTS: 18 eyes of 18 patients were included. All patients underwent a DMEK with undersized graft after failed PK(s). Mean time between the last PK and DMEK was 102 ± 82 weeks. Mean follow-up time was 8.9 ± 4.6 months. CDVA increased significantly from 1.12 ± 0.60 logMAR preoperatively to 0.64 ± 0.49 logMAR 6 weeks postoperatively (p = 0.013). Mean CCT decreased significantly from 807 ± 224 µm before to 573 ± 151 µm 6 weeks after DMEK (p = 0.003). Re-bubbling was necessary in eight eyes (44.4%) after a median time of 7 days. The 12-month Kaplan Meier survival was 66.7%. CONCLUSION: In case of endothelial graft decompensation without stromal scars after primary PK, a DMEK can be performed for selected patients who had satisfying CDVA before the endothelial decompensation. Prior to DMEK indication, an AS-OCT should routinely be performed to circularly search for posterior steps at the PK graft margin, as well as shortly after DMEK to exclude a detachment of the endothelial graft. All patients should be informed about a higher re-bubbling rate in comparison to primary DMEK.

In situ donor keratometry in deceased patients as a novel screening technique for eye banking.

PURPOSE: To investigate the potential role of keratometry on whole globes in situ of deceased patients by assessing its repeatability and comparing it with sterile donor tomography after excision and preservation in organ culture. METHODS: A sequence of 5 measurements was taken from 40 eyes in situ of deceased patients < 24 h after death using the portable Retinomax K-plus 3 (Bon, Tokyo, Japan). Keratometry of whole globes in situ, from which sclerocorneal discs were taken for organ culture, was compared to those obtained after measuring these sclerocorneal disks through their cell culture flask in medium I after 5 ± 4 days using the anterior segment optical coherence tomograph Casia 2 (Tomey Corp., Nagoya, Japan), and to 964 different donor corneas in medium II. RESULTS: Cronbach's alpha of the in situ keratometry was 0.891 and 0.942 for the steepest and flattest corneal power (P). The steepest (44.5D) and flattest (41.1D) P as well as the astigmatism (3.4D) of in situ corneas remained unchanged after preserving sclerocorneal discs in medium I (respectively 44.7D [p = 0.09]; 41.4D [p = 0.17]; 3.3D [p = 0.09]). The comparison of the in situ values with the 964 measured different donor corneas in medium II showed significantly (p < 0.001) higher P at the steep (45.4D) and flat (43.9D) meridian and smaller astigmatism (1.4D) for sterile donor tomography. CONCLUSIONS: Measuring deceased patients' eyes in situ with the portable Retinomax K-plus 3 represents a feasible and reliably repeatable screening method in the eye bank. In comparison to donor tomography in medium I, it measures a similar power and astigmatism.

YB-1 unwinds mRNA secondary structures in vitro and negatively regulates stress granule assembly in HeLa cells.

In the absence of the scanning ribosomes that unwind mRNA coding sequences and 5'UTRs, mRNAs are likely to form secondary structures and intermolecular bridges. Intermolecular base pairing of non polysomal mRNAs is involved in stress granule (SG) assembly when the pool of mRNAs freed from ribosomes increases during cellular stress. Here, we unravel the structural mechanisms by which a major partner of dormant mRNAs, YB-1 (YBX1), unwinds mRNA secondary structures without ATP consumption by using its conserved cold-shock domain to destabilize RNA stem/loops and its unstructured C-terminal domain to secure RNA unwinding. At endogenous levels, YB-1 facilitates SG disassembly during arsenite stress recovery. In addition, overexpression of wild-type YB-1 and to a lesser extent unwinding-defective mutants inhibit SG assembly in HeLa cells. Through its mRNA-unwinding activity, YB-1 may thus inhibit SG assembly in cancer cells and package dormant mRNA in an unfolded state, thus preparing mRNAs for translation initiation.

Baseline Characteristics of 1976 Patients With Ectatic Corneal Disorders at a Single Center From 2010 to 2021: A Cross-Sectional Study of the Homburg Keratoconus Center.

OBJECTIVES: The study objective was to analyze the baseline characteristics of keratoconus (KC) patients at the Homburg Keratoconus Center from 2010 to 2021. METHODS: This cross-sectional study included 3,674 eyes, with analysis of demographics, clinical findings, visual function, endothelial measurements, and topographic, tomographic, and corneal biomechanical data from the first visit. RESULTS: Mean patient age was 36.3±13.8 years. The mean uncorrected distance visual acuity in log of minimal angle of resolution was 0.60 (20/80, Snellen equivalent), and the corrected mean was 0.3 (20/40). Of 1976 patients, 48.9% reported eye rubbing. Mean values (ranges) were 49.4±6.3 (36.3-78.0) D for steep keratometry, 462.4±66.0 (48.0-659.0) µm for thinnest corneal thickness, 9.7±8.7 (-0.5 to 88.8) for Belin/Ambrósio enhanced ectasia total deviation, 0.8±0.4 (0.0-1.0) for the Corvis biomechanical index, 0.9±0.2 (0.0-1.0) for the tomographic biomechanical index, 0.1±0.5 (-0.9 to 2.0) for the KC match index, 8.3±1.8 (2.2-17.7) mm Hg for corneal hysteresis, 7.1±2.2 (0.0-17.0) mm Hg for corneal resistance factor, and 2,562.9±326.3 (1,011-3,937) cells/mm2 for endothelial cell density. The average ABCDE KC stage was A2B3C1D1E2. Distance-corrected visual acuity correlated strongly with topometric, tomographic, and biomechanical data ( P <0.001). CONCLUSIONS: This comprehensive description of baseline features of KC patients at a tertiary center provides a reference for further longitudinal and international multicentric studies.

Ultrastructural Examination of the Corneal Interface after Predescemetic Deep Anterior Lamellar Keratoplasty (DALK) - A Case Report with Light and Transmission Electron Microscopy.

PURPOSE: To examine corneal buttons with light and transmission electron microscopy (TEM) to visualize the interface area and highlight the ultrastructural corneal changes after deep anterior lamellar keratoplasty (DALK). METHODS: Two patients underwent excimer laser-assisted penetrating repeat keratoplasty after predescemetic DALK. The corneal buttons were examined by light microscopy and TEM. RESULTS: The light microscopic examination of the corneal buttons revealed fragments of a second Descemet's membrane in the central and midperipheral areas (Case 1). In both cases, visualization of the interface area was not possible by light microscopy. The donor and host stroma were tightly attached without dehiscence. TEM identified the interface area by irregularities in the collagen distribution between the donor and host stroma. The thickness of the remaining recipient corneal stroma measured approximately 30 µm (Case 1) and 100 µm (Case 2), respectively. In the host stroma, TEM revealed the absence or degeneration of keratocytes, accumulation of amorphous material between the collagen lamellae, and vacuolar inclusions dispersed in the stroma, forming a band-like zone anterior to Descemet's membrane. CONCLUSION: The interface area after DALK has been mainly investigated by in vivo confocal microscopy. Light microscopy and TEM findings indicate remodeling processes after DALK that are associated with increased keratocyte degeneration and structural alterations of the extracellular matrix in the host stroma. The choice of surgical method may influence the postoperative morphological and functional outcome since these findings were primarily apparent in the remaining host stroma. Therefore, complete exposure of Descemet's membrane is an important prognostic factor for the postoperative visual outcome.

Risk Factors for Keratoconus Progression in Children Compared with Young and Middle-aged Adults.

BACKGROUND: To investigate the risk factors for keratoconus progression in children (10 - 18 years old; age group 1) compared to young adults (19 - 35 years old; age group 2) and middle-aged adults (36 - 55 years old; age group 3). PATIENTS/METHODS: Ninety-seven children, 445 young adults, and 342 middle-aged adults underwent total ophthalmic examination including clinical refraction, slit lamp examination, corneal tomography, eye biometry, and biomechanical properties measurements. Comparisons were assessed among three age groups and between progressive and nonprogressive eyes. Logistic regression was applied to determine the potential prognostic factors for keratoconus progression in the three age groups. RESULTS: Univariate logistic regression analysis show that the most prominent factors associated with progression were corneal posterior vertical radius (RVP), eye rubbing (RUB), slit lamp corneal thinning (SLT), contact lens use (CL), and central corneal thickness (CCT) in all age groups. Additionally, the anterior chamber volume (ACV) and keratoconus match index (KMI) were associated with progression in age group 1. Location of the thinnest corneal thickness at the vertical axis (TCTy), distance from apex to the thinnest point (BADISTAPEX), scissor reflection in retinoscopy (SKIAREFLEX), and Vogt striae were associated with progression in age group 2, and TCTy, anterior and posterior asphericity (ASPA and ASPP, respectively), BADISTAPEX, SKIAREFLEX, and Vogt striae were associated with progression in age group 3. The multivariate model with the highest predictability indicated RVP, ACV, and SLT as independent determinants of progression in age group 1 (AUC: 90%, sensitivity: 88.9%, specificity: 90.9%), RVP, ACV, SLT, and SKIAREFLEX in group 2 (AUC: 81.6%, sensitivity 88.5%, specificity: 70.3%), and RVP, SLT, Vogt striae, and CL in age group 3 (AUC: 80%, sensitivity 82.8%, specificity: 73%). CONCLUSION: ACV and KMI seem to play a major role in the progression of pediatric KC compared to adults. This is probably due to different anatomical and biomechanical characteristics of a child's eye globe.

YB-1 Structure/Function Relationship in the Packaging of mRNPs and Consequences for Translation Regulation and Stress Granule Assembly in Cells.

From their synthesis in the nucleus to their degradation in the cytoplasm, all mRNAs have the same objective, which is to translate the DNA-stored genetic information into functional proteins at the proper time and location. To this end, many proteins are generally associated with mRNAs as soon as transcription takes place in the nucleus to organize spatiotemporal regulation of the gene expression in cells. Here we reviewed how YB-1 (YBX1 gene), one of the major mRNA-binding proteins in the cytoplasm, packaged mRNAs into either compact or extended linear nucleoprotein mRNPs. Interestingly the structural plasticity of mRNPs coordinated by YB-1 could provide means for the contextual regulation of mRNA translation. Posttranslational modification of YB-1, notably in the long unstructured YB-1 C-terminal domain (CTD), and/or the protein partners of YB-1 may play a key role in activation/inactivation of mRNPs in the cells notably in response to cellular stress.

Morphological characterization and clinical effects of stromal alterations after intracorneal ring segment implantation in keratoconus.

PURPOSE: To analyze the histological and (ultra)structural stromal tissue changes after femtosecond (Fs) laser-assisted intracorneal ring segment (ICRS) implantation and their refractive and topographic effects in patients with keratoconus. METHODS: This monocentric retrospective case series included 15 consecutive patients with clinical peri-segmental lamellar channel deposits after treatment with Fs-ICRS implantation for keratoconus. The stromal changes were investigated using in vivo confocal microscopy. Two patients underwent a penetrating keratoplasty after the Fs-ICRS implantation; the explanted corneas were processed for histopathology and transmission electron microscopy (TEM). Refractive and topographic effects were investigated comparing the uncorrected (UDVA) and corrected (CDVA) distance visual acuity, spherical equivalent (SE), flat (K1), steep (K2), and steepest (Kmax) keratometry before and after detection of lamellar channel deposits. RESULTS: In vivo confocal microscopy revealed diffuse linear and focal granular hyperreflective structures. Histologically, there was mild proliferation of fibroblasts and fibrosis. TEM demonstrated focal accumulations of degenerated keratocytes with cytoplasmic lipid inclusions. There were no significant changes for UDVA (Δ = 0.0 ± 0.2 logMAR; p = 0.67), CDVA (Δ = 0.0 ± 0.1 logMAR; p = 0.32), SE (Δ 0.1 ± 0.9 D; p = 0.22), K1 (Δ = 0.3 ± 1.0 D; p = 0.28), K2 (Δ = 0.1 ± 0.9 D; p = 0.51), and Kmax (Δ = 0.3 ± 1.5 D; p = 0.17). CONCLUSIONS: Two types of structural stromal changes were identified: (1) diffuse peri-segmental fibrosis and (2) lamellar channel deposits. These structural changes showed no evidence of a relevant refractive or topographic effect.

Approval rates for corneal donation and the origin of donor tissue for transplantation at a university-based tertiary referral center with corneal subspecialization hosting a LIONS Eye Bank.

BACKGROUND: With the increasing demand for corneas, eye banks must optimize the tissue donation, collection, and selection process. This retrospective monocentric study analyzed the approval rates for corneal donation and the origin of and reasons for discarding donor corneas from 2010 to 2019. METHODS: Data included the number of deceased, approval or rejection by the family for corneal donation and contraindications. Corneal grafts were included from all deceased persons who were full-body and multi-organ donors at the Saarland University Medical Center (UKS) and from external institutions. Additional analyzed parameters included endothelial cell count (ECC), blood sample serology for infections, and conjunctival swab testing . RESULTS: A total of 1748 corneoscleral buttons were harvested from 10,265 deceased persons (17% with no contraindication) at the UKS between 2010 and 2019, with a consent rate of 23.3%. The number of keratoplasties increased from 136 in 2010 (15% of the deceased, total = 925) to 251 in 2019 (21%, total = 1214). Both the general and department-specific data showed similar percentages for corneal donation over the years, with intensive care and palliative units recently providing the most corneas. The increase in the number of corneas processed by the cornea bank over the years (368 in 2010 compared with 857 in 2019) was linked both to a better internal supply in 2010 (262, 71.2% of the total) compared with 2019 (519, 60.6%) and to an external supply by reinforcement of cooperation with external hospitals, including Luxembourg in 2010 (106, 28.8% of the total) compared with 2019 (338, 39.4%). A total of 195 of 377 corneas (52%) were discarded in 2009 compared with 260 out of 715 (36%) in 2019. The main reasons for discarding were low ECC (36% of discarded corneas in 2009; 11% in 2019), positive conjunctival swab (11% in 2009; 13% in 2019), and blood sample serology (6% in 2009 and in 2019). CONCLUSION: Despite an increasing number of donors, the demand for corneas is still rising. Improved cooperation with internal departments and with external clinics has led to an increasing number of explanted corneas. The main reason for discarding corneas was low ECC, followed by a positive conjunctival swab for fungal or bacterial contamination and serology. Increased donation rates and continued improvements in collection and selection processes are necessary to cover the high demand for corneas.

Corneal epithelial ingrowth after perforating corneal injury: a case report.

BACKGROUND: Epithelial ingrowth is a rare complication after ocular perforation and can become manifest many years after the primary trauma. CASE PRESENTATION: A 49-year-old patient presented with a positive Seidel test of unclear origin at her left eye, as well as a sharply defined anterior-stromal corneal scar at both eyes. Prior operations included a bilateral laser-assisted blepharoplasty 3 months earlier. The patient indicated to have been on holiday to France 5 months earlier, during an ongoing oak processionary moth caterpillars infestation. The examination using confocal microscopy confirmed a corneal perforation at the left eye and revealed corneal epithelial ingrowth capped with scarred stroma in both eyes. We performed a penetrating keratoplasty at the left eye. The scarred and perforated host cornea was divided into 4 pieces for further investigation: microbiology (negative), virology (negative), histology and transmission electron microscopy (TEM). Histology revealed differently structured epithelium, centrally inverted into the stroma through defects in Bowman's layer. TEM revealed full thickness corneal perforation with an epithelial plug extending to the lower third of the cornea, but without evidence of epithelial cell migration into the anterior chamber. Our differential diagnosis of the unclear positive Seidel test with epithelial ingrowth was as follows: (1) corneal perforation by hairs of the oak processionary moth caterpillar, although no hairs could be found histologically; (2) corneal perforation during laser-assisted blepharoplasty, which may be supported by the presence of pigmented cells on the posterior surface of Descemet´s membrane, pointing to a possible iris injury. CONCLUSION: Consequently, we highlighted that contact lenses can be useful, safe and inexpensive protective devices in upper eyelid procedures to protect the cornea against mechanical iatrogenic trauma.

Reliability and efficiency of corneal thickness measurements using sterile donor tomography in the eye bank.

To evaluate the reliability and efficiency of sterile pachymetric measurements of donor corneas based on tomographic data using two different methods: a "manual" and a "(semi-)automated" method. Twenty-five (25) donor corneas (50%) stored in MI and 25 (50%) in MII were imaged 5 times consecutively using an anterior segment OCT (AS-OCT). The central corneal thickness (CCT) was measured both with the manual measurement tool of the AS-OCT (= CCTm) and with a MATLAB self-programmed software allowing (semi-)automated analysis (= CCTa). We analyzed the reliability of CCTm and CCTa using Cronbach´s alpha (α) and Wilcoxon signed-Rank Test. Concerning CCTm, 68 measurements (54.4%) in MI and 46 (36.8%) in MII presented distortions in the imaged 3D-volumes and were discarded. Concerning CCTa, 5 (4%) in MI and 1 (0.8%) in MII were not analyzable. The mean (± SD) CCTm was 1129 ± 6.8 in MI and 820 ± 5.1 µm in MII. The mean CCTa was 1149 ± 2.7 and 811 ± 2.4 µm, respectively. Both methods showed a high reliability with a Cronbach´s α for CCTm of 1.0 (MI/MII) and for CCTa of 0.99 (MI) and 1.0 (MII). Nevertheless, the mean SD of the 5 measurements was significantly higher for CCTm compared to CCTa in MI (p = 0.03), but not in MII (p = 0.92). Sterile donor tomography proves to be highly reliable for assessment of CCT with both methods. However, due to frequent distortions regarding the manual method, the (semi-)automated method is more efficient and should be preferred.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

OCT Application for Sterile Corneal Graft Screening in the Eye Bank. / OCT-Einsatz für das sterile Spender-Screening in der Hornhautbank.

BACKGROUND AND OBJECTIVE: Sterile donor tomography enables the detection of corneal tissues with refractive anomalies. The aim of this study was to determine the curvature and thickness of donor corneas to support proper selection in the eye bank. METHODS: 704 donor corneas (Klaus Faber Center, LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, in Homburg/Saar) were measured using the anterior segment optical coherence tomograph (AS-OCT) CASIA 2 (Tomey Corp., Nagoya, Japan). The corneoscleral discs were measured in their cell culture flask, which was positioned in a holder on the chin rest of the AS-OCT, after conversion to medium II (with 6% dextran T-500). The measured raw data were analysed and processed in MATLAB (MathWorks Inc., Natick, Massachusetts, USA), after which the refractive power of the steep and flat meridian at the anterior and posterior surface and the central corneal thickness (CCT) of the donor corneas were determined. Results values are expressed as mean x̅ ± standard deviation SD. RESULTS: The mean refractive power of the steep/flat meridian at the anterior surface was 45.4 ± 1.8 D/44.0 ± 1.3 D, the corresponding values for the posterior surface were - 6.2 ± 0.3 D/- 5.9 ± 0.2 D, and the mean CCT was 616.3 ± 85.1 µm. Of the 704 (100%) measured donor tissues, 590 (83.8%)/670 (95.2%) donor corneas showed no anomaly beyond respectively x̅ ± 2 SD/x̅ ± 3 SD among the 5 examined parameters. 72 (10.3%)/23 (3.3%) donor corneas had only 1 anomaly, 26 (3.7%)/10 (1.4%) had 2 anomalies, 10 (1.4%)/1 (0.1%), 3 anomalies, 5 (0.7%)/0 (0.0%), 4 anomalies, and 1 (0.1%)/0 (0.0%), 5 anomalies. CONCLUSIONS: AS-OCT provides an objective and sterile screening method to identify corneal tissues with curvature anomalies in order to further optimise donor selection in the eye bank. To avoid postoperative refractive surprises, donor corneas with a total refractive power that deviates > ± 3 SD from the mean should not be used for penetrating or anterior lamellar keratoplasty, but may be suitable for posterior lamellar keratoplasty (DMEK or DSAEK). In the future, sterile donor tomography could enable: (1) the harmonisation of donor and recipient tomography, which may minimise residual astigmatism for a particular donor-recipient pair; and (2) the improvement of IOL power calculation in a classical triple procedure by means of regression analysis between pre- and postoperative total refractive power of corneal grafts.

Microtubules as platforms for probing liquid-liquid phase separation in cells - application to RNA-binding proteins.

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.

Relation Between Stress Granules and Cytoplasmic Protein Aggregates Linked to Neurodegenerative Diseases.

ᅟ: A hallmark of neurodegenerative diseases is the accumulation of cytoplasmic protein aggregates in neurons of affected subjects. Among recently identified elements of these aggregates are RNA-binding proteins (RBPs) involved in RNA metabolism and alternative splicing and have in common the presence of low complexity domains (LCD) that are prone to self-assemble and form aggregates. The mechanism of cytoplasmic protein aggregation remains elusive. Stress granules (SGs) that are micrometric RNA-protein assemblies located in the cytoplasm of cells exposed to environmental stress are suspected to play the role of seeds. The review sheds light on the recent experimental results that suggest a link between SGs and cytoplasmic protein aggregates but also propose other routes for the formation of these aggregates. PURPOSE OF REVIEW: To analyze the potential relationship between cytoplasmic protein aggregates in neurons of affected subjects and stress granules. RECENT FINDINGS: Liquid phase separation explains how protein and RNA could assemble in membraneless compartments, notably SGs. These results highlight the importance of RBPs with LCD in the SG assembly. Maturation of SGs and in particular the dense core is a potential source of insoluble protein aggregates. Several lines of evidence linked stress granule dynamics to pathogenic protein aggregates. (i) Proteins that accumulate in cytoplasmic aggregates are also SG components. (ii) Neurons are specifically exposed to stress events due to their high metabolism and long lifespan. (iii) Diseases linked protein mutations affect the SG dynamics. (iv) SG dense core could be a breeding ground for protein aggregates. However, we should also keep in mind that SGs are not the only RNA-protein assembly in the cytoplasm; the RNA transport granules could also play a role in the formation of insoluble protein aggregates.

Polyamine signal through gap junctions: A key regulator of proliferation and gap-junction organization in mammalian tissues?

We propose that interaction rules derived from polyamine exchange in connected cells may explain the spatio-temporal organization of gap junctions observed during tissue regeneration and tumorigenesis. We also hypothesize that polyamine exchange can be considered as signal that allows cells to sense the proliferation status of their neighbors. Polyamines (putrescine, spermidine, and spermine) are indeed small aliphatic polycations that serve as fuels to sustain elevated proliferation rates of the order observed in cancer cells. Based on recent reports, we consider here that polyamines can be exchanged through gap junction channels between mammalian cells. Such intercellular exchange of polyamines has critical consequences on the local control of growth. In line with this hypothesis, the complex protein network that keeps polyamine levels finely tuned in mammalian cells can translate polyamine efflux or influx into integrated signals controlling transcription, translation, and cell communications.

Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.

Role of tau in the spatial organization of axonal microtubules: keeping parallel microtubules evenly distributed despite macromolecular crowding.

Opposing views have been proposed regarding the role of tau, the principal microtubule-associated protein in axons. On the one hand, tau forms cross-bridges at the interface between microtubules and induces microtubule bundling in neurons. On the other hand, tau is also considered a polymer brush which efficiently separates microtubules. In mature axons, microtubules are indeed arranged in parallel arrays and are well separated from each other. To reconcile these views, we developed a mechanistic model based on in vitro and cellular approaches combined to analytical and numerical analyses. The results indicate that tau forms long-range cross-bridges between microtubules under macromolecular crowding conditions. Tau cross-bridges prevent the redistribution of tau away from the interface between microtubules, which would have occurred in the polymer brush model. Consequently, the short-range attractive force between microtubules induced by macromolecular crowding is avoided and thus microtubules remain well separated from each other. Interestingly, in this unified model, tau diffusion on microtubules enables to keep microtubules evenly distributed in axonal sections at low tau levels.