ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes.

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes.

RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.

Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes.

Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.

Vigilin interacts with signal peptide peptidase.

BACKGROUND: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. RESULTS: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. CONCLUSIONS: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.

Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes.

The Wellcome Trust Case Control Consortium (WTCCC) primary genome-wide association (GWA) scan on seven diseases, including the multifactorial autoimmune disease type 1 diabetes (T1D), shows associations at P < 5 x 10(-7) between T1D and six chromosome regions: 12q24, 12q13, 16p13, 18p11, 12p13 and 4q27. Here, we attempted to validate these and six other top findings in 4,000 individuals with T1D, 5,000 controls and 2,997 family trios independent of the WTCCC study. We confirmed unequivocally the associations of 12q24, 12q13, 16p13 and 18p11 (P(follow-up)

Hemodynamic effects of the vacuum-assisted closure device on open mediastinal wounds.

BACKGROUND: Application of the Vacuum-Assisted Closure device (VAC) to open sternal wounds has negative hemodynamic effects. We hypothesized that the interposition of a muscle flap attenuates these negative hemodynamic effects. MATERIALS AND METHODS: After institutional approval, monitoring lines were placed in anesthetized, ventilated pigs. Through a median sternotomy, sonometric crystals were strategically positioned around the left ventricle. A rectus flap was rotated over the mediastinal wound, and the VAC was placed over the flap. After baseline measurements, a vacuum of 125 mmHg [Group (GP) 1, n = 5] or 50 mmHg (GP2, n = 6) was initiated. Hemodynamics were recorded every 15 min for 1.5 h, and 15 min after cessation of the vacuum therapy. GP3 (n = 6) underwent intermittent VAC cycling (on 5 min/off 2 min). Significance determined by t test. RESULTS: While non-flapped animals had significant detriment in both left ventricular filling volume and cardiac output, flapped animals had insignificant depression of both parameters. CONCLUSION: Application of muscle flaps to sternal wounds prior to VAC therapy significantly attenuates the negative hemodynamic effects seen when the VAC is used alone.

Speech benefits of posterior pharyngeal flap are preserved after surgical flap division for obstructive sleep apnea: experience with division of 12 flaps.

Attachment of a posterior pharyngeal flap is commonly performed for the surgical management of velopharyngeal insufficiency. Obstructive sleep apnea has been found to occur in as many as 38% of patients undergoing posterior pharyngeal flap attachment. Often, this is temporary in the early postoperative period. When it occurs later after surgery, however, it can require active treatment. Many patients improve with the use of nighttime nasal C-PAP. Those patients who do not improve sufficiently with nasal C-PAP may require surgical flap division. We report surgical flap division in 12 such patients. All patients had preoperative and postoperative perceptual speech evaluations, and most had preoperative and postoperative pressure flow studies. In 11 of the 12 patients who underwent surgical flap division, velopharyngeal function did not deteriorate. We hypothesized that the persistence of the speech improvement in those patients is either secondary to the bulk of tissue from the flap, which remains in the posterior pharyngeal wall and provides a pad to assist with velopharyngeal closure, or is secondary to the speech mechanisms that the patients learned with the flap in place and were able to continue even after flap division.

Laparoscopic omental harvest for flap coverage in complex mediastinitis.

Mediastinitis is one of the most serious complications of cardiac surgery. The standard of care in mediastinitis includes thorough sequential debridement, flap coverage, and culture-directed antibiotics. The most frequently utilized muscles for flap reconstruction include the rectus abdominus and the pectoralis major. However, in some instances these flaps may be inadequate, unavailable, or fail, thus requiring an alternative choice or adjuvant. Most coronary graft procedures utilize the left internal mammary artery, frequently eliminating the left rectus muscles, while prior open cholecystectomy patients frequently lose availability of their right rectus muscle. In addition, radiation therapy or prior flap failure may exclude other muscle transfer procedures. The omentum offers excellent coverage due to mobility and superb arterial and lymphatic flow. Unfortunately, in the past, this has required a celiotomy in an already critically ill patient. We present a series of 5 patients where the omentum was mobilized laparoscopically and passed through an anterior diaphragmatic incision. This option spares a celiotomy, seals the wound, and hastens recovery in very ill patients. We also present a complete review of literature on the topic and provide an algorithm for complex sternal wound reconstruction.

Innovative techniques in bony reconstruction to facilitate hand salvage.

Mutilating injuries of the hand and congenital hand anomalies can present challenging reconstructive scenarios for salvage and restoration of function. During a 5-year period from 1993 to 1997, the plastic and reconstructive surgical unit of East Carolina University Medical Center was presented with a series of unique reconstructive challenges as a result of complex hand injuries that resulted in unexpected opportunities for the salvage of distal components. These traumatic injuries were unique in that, although devastating to the hand, they left the opportunity for salvage of distal vascularized and sensate components of the hand. Other unique challenges arose as a result of patients who did not want to pursue alternative reconstructive options such as toe-to-hand transfers or pollicization. These cases are presented to emphasize alternative algorithms to standard hand reconstruction in complex scenarios. Three patients presented with distal viable (vascularized and sensate) phalangeal components with proximal complex bony defects, 1 patient presented with a complex thumb defect and declined standard therapy, and 1 patient presented with a congenital thumb anomaly and declined standard therapy. All flaps survived and all hands were saved. These patients illustrate the clinical feasibility of osteocutaneous and free osseous grafting to provide strut stabilization in metacarpal defects and to preserve an opposable post after thumb amputation or thumb anomaly.