Woronin body hitchhiking on early endosomes is dispensable for septal localization in Aspergillus nidulans.

The proper functioning of organelles depends on their intracellular localization, mediated by motor protein-dependent transport on cytoskeletal tracks. Rather than directly associating with a motor protein, peroxisomes move by hitchhiking on motile early endosomes in the filamentous fungus Aspergillus nidulans. However, the physiological role of peroxisome hitchhiking is unclear. Peroxisome hitchhiking requires the protein PxdA, which is conserved within the fungal subphylum Pezizomycotina but absent from other fungal clades. Woronin bodies are specialized peroxisomes that are also unique to the Pezizomycotina. In these fungi, multinucleate hyphal segments are separated by incomplete cell walls called septa that possess a central pore enabling cytoplasmic exchange. Upon damage to a hyphal segment, Woronin bodies plug septal pores to prevent widespread leakage. Here, we tested whether peroxisome hitchhiking is important for Woronin body motility, distribution, and function in A. nidulans. We show that Woronin body proteins are present within all motile peroxisomes and hitchhike on PxdA-labeled early endosomes during bidirectional, long-distance movements. Loss of peroxisome hitchhiking significantly affected Woronin body distribution and motility in the cytoplasm, but Woronin body hitchhiking is ultimately dispensable for septal localization and plugging.

Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes in Aspergillus nidulans.

The proper functioning of organelles depends on their intracellular localization, mediated by motor protein-dependent transport on cytoskeletal tracks. Rather than directly associating with a motor protein, peroxisomes move by hitchhiking on motile early endosomes in the filamentous fungus Aspergillus nidulans . However, the cellular function of peroxisome hitchhiking is unclear. Peroxisome hitchhiking requires the protein PxdA, which is conserved within the fungal subphylum Pezizomycotina, but absent from other fungal clades. Woronin bodies are specialized peroxisomes that are also unique to the Pezizomycotina. In these fungi, multinucleate hyphal segments are separated by incomplete cell walls called septa that possess a central pore enabling cytoplasmic exchange. Upon damage to a hyphal segment, Woronin bodies plug septal pores to prevent wide-spread leakage. Here, we tested if peroxisome hitchhiking is important for Woronin body motility, distribution, and function in A. nidulans . We show that Woronin body proteins are present within all motile peroxisomes and hitchhike on PxdA-labeled early endosomes during bidirectional, long-distance movements. Loss of peroxisome hitchhiking by knocking out pxdA significantly affected Woronin body distribution and motility in the cytoplasm, but Woronin body hitchhiking is ultimately dispensable for septal localization and plugging.

Hitchhiking Across Kingdoms: Cotransport of Cargos in Fungal, Animal, and Plant Cells.

Eukaryotic cells across the tree of life organize their subcellular components via intracellular transport mechanisms. In canonical transport, myosin, kinesin, and dynein motor proteins interact with cargos via adaptor proteins and move along filamentous actin or microtubule tracks. In contrast to this canonical mode, hitchhiking is a newly discovered mode of intracellular transport in which a cargo attaches itself to an already-motile cargo rather than directly associating with a motor protein itself. Many cargos including messenger RNAs, protein complexes, and organelles hitchhike on membrane-bound cargos. Hitchhiking-like behaviors have been shown to impact cellular processes including local protein translation, long-distance signaling, and organelle network reorganization. Here, we review instances of cargo hitchhiking in fungal, animal, and plant cells and discuss the potential cellular and evolutionary importance of hitchhiking in these different contexts.

Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS-Hook-FHIP complexes.

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS-Hook-FHIP ('FHF') cargo adaptor complex links dynein to cargo in humans and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein 'interactome' of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 colocalize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A, and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS-Hook-FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

Optimizing microtubule arrangements for rapid cargo capture.

Cellular functions such as autophagy, cell signaling, and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow undirected movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus ends relative to the initial cargo position. We use analytic mean first-passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule arrangements that minimize mean cargo capture times. Namely, we show that configurations with a single microtubule plus end abutting the distal tip and broadly distributed other plus ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between the distribution of microtubule tips and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

PxdA interacts with the DipA phosphatase to regulate peroxisome hitchhiking on early endosomes.

In canonical microtubule-based transport, adaptor proteins link cargoes to dynein and kinesin motors. Recently, an alternative mode of transport known as "hitchhiking" was discovered, where cargoes achieve motility by hitching a ride on already-motile cargoes, rather than attaching to a motor protein. Hitchhiking has been best studied in two filamentous fungi, Aspergillus nidulans and Ustilago maydis. In U. maydis, ribonucleoprotein complexes, peroxisomes, lipid droplets (LDs), and endoplasmic reticulum hitchhike on early endosomes (EEs). In A. nidulans, peroxisomes hitchhike using a putative molecular linker, peroxisome distribution mutant A (PxdA), which associates with EEs. However, whether other organelles use PxdA to hitchhike on EEs is unclear, as are the molecular mechanisms that regulate hitchhiking. Here we find that the proper distribution of LDs, mitochondria, and preautophagosomes do not require PxdA, suggesting that PxdA is a peroxisome-specific molecular linker. We identify two new pxdA alleles, including a point mutation (R2044P) that disrupts PxdA's ability to associate with EEs and reduces peroxisome movement. We also identify a novel regulator of peroxisome hitchhiking, the phosphatase DipA. DipA colocalizes with EEs and its association with EEs relies on PxdA. Together, our data suggest that PxdA and the DipA phosphatase are specific regulators of peroxisome hitchhiking on EEs.

Topical Vitamin D May Modulate Human Sinonasal Mucosal Responses to House Dust Mite Antigen.

BACKGROUND: Respiratory epithelium is a key defense against inhaled pathogens. Vitamin D3 (VD) has been suggested to modulate airway inflammation; however, its effect on innate airway defenses, the physical barrier, mucociliary apparatus, and cytokine release remains unclear. OBJECTIVE: To investigate the outcomes of VD application prior to challenge in an in vitro model of human sinonasal epithelium, through assessment of epithelial transepithelial resistance (TER), cilia beat frequency (CBF), and interleukin (IL)-6 release, and secondarily to determine whether topical VD is beneficial to patients with inflammatory sinonasal pathology. METHODS: Primary human sinonasal epithelial cells from patients with eosinophilic chronic rhinosinusitis (eCRS) and healthy controls were cultured in air-liquid interface (ALI). Well-differentiated cultures from each patient were pretreated for 24 hours with 4 different VD doses. Toxicity was quantified at 24 hours in unchallenged ALI by lactate dehydrogenase (LDH) assay. Innate responses were assessed by measuring TER and CBF before and up to 24 hours after house dust mite Dermatophagoides pteronyssinus challenge. IL-6 release was evaluated 24-hour postchallenge. RESULTS: Fifteen patients (53 ± 13.5 years, 60% females, 53% eCRS) representing 120 ALI wells were assessed. VD (0, 25, 50, 150 IU/mL) released less LDH than vehicle, indicating noncytotoxicity (0.15 ± 0.02; 0.15 ± 0.00; 0.14 ± 0.02; 0.11 ± 0.01 vs 0.17 ± 0.03, P = .004). VD increased TER for eCRS wells at 5 minutes (50 IU/mL: Δ6.76 ± 3.93 vs Δ3.87 ± 2.46, P = .04) and 24 hours (50 IU/mL: Δ0.88 ± 0.49 vs Δ0.40 ± 0.42, P = .02; 150 IU/mL: Δ1.06 ± 0.58 vs Δ0.47 ± 0.46, P = .01). CBF increased at 1 hour for eCRS wells (50 IU/mL: Δ0.62 ± 0.14 vs Δ0.41 ± 0.13, P = .001; 150 IU/ml: Δ0.60 ± 0.13 vs Δ0.38 ± 0.11, P < .001). IL-6 release was similar between normal and eCRS wells. CONCLUSION: Topical VD supplementation in eCRS patients may be beneficial for innate epithelial defenses. VD is noncytotoxic and does not adversely affect the physical barrier, mucociliary apparatus, or IL-6 release. Further studies should clarify its potential as a therapeutic agent.

Hitching a Ride: Mechanics of Transport Initiation through Linker-Mediated Hitchhiking.

In contrast to the canonical picture of transport by direct attachment to motor proteins, recent evidence shows that a number of intracellular "cargos" navigate the cytoplasm by hitchhiking on motor-driven "carrier" organelles. We describe a quantitative model of intracellular cargo transport via hitchhiking, examining the efficiency of hitchhiking initiation as a function of geometric and mechanical parameters. We focus specifically on the parameter regime relevant to the hitchhiking motion of peroxisome organelles in fungal hyphae. Our work predicts the dependence of transport initiation rates on the distribution of cytoskeletal tracks and carrier organelles, as well as the number, length, and flexibility of the linker proteins that mediate contact between the carrier and the hitchhiking cargo. Furthermore, we demonstrate that attaching organelles to microtubules can result in a substantial enhancement of the hitchhiking initiation rate in tubular geometries such as those found in fungal hyphae. This enhancement is expected to increase the overall transport rate of hitchhiking organelles and lead to greater efficiency in organelle dispersion. Our results leverage a quantitative physical model to highlight the importance of organelle encounter dynamics in noncanonical intracellular transport.

Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed-end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.

Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast.

We previously discovered that competition between fission yeast actin binding proteins (ABPs) for binding F-actin facilitates their sorting to different cellular networks. Specifically, competition between endocytic actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances their activities, and prevents tropomyosin Cdc8's association with actin patches. However, these interactions do not explain how Fim1 is prevented from associating strongly with other F-actin networks such as the contractile ring. Here, we identified α-actinin Ain1, a contractile ring ABP, as another Fim1 competitor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their F-actin residence time. While Fim1 outcompetes both Ain1 and Cdc8 individually, Cdc8 enhances the F-actin bundling activity of Ain1, allowing Ain1 to generate F-actin bundles that Cdc8 can bind in the presence of Fim1. Therefore, the combination of contractile ring ABPs Ain1 and Cdc8 is capable of inhibiting Fim1's association with F-actin networks.

Association Between Mental Health Status and Patient Satisfaction With the Functional Outcomes of Rhinoplasty.

IMPORTANCE: Mental health can have an impact on patient satisfaction with rhinoplasty. However, the association between mental health and patient satisfaction with functional outcomes of rhinoplasty is poorly understood. OBJECTIVE: To determine whether preoperative mental health is associated with satisfaction with functional outcomes of rhinoplasty. DESIGN, SETTING, AND PARTICIPANTS: This case-control study assessed baseline nasal function and postsurgical functional outcomes for 88 consecutive patients undergoing rhinoplasty with both cosmetic and functional goals at 2 tertiary rhinologic centers in Sydney, Australia. EXPOSURES: Poor mental well-being was defined preoperatively by the Optum SF-36v2 Health Survey mental component summary. MAIN OUTCOMES AND MEASURES: Nasal function was assessed with patient-reported outcome measures, including visual analog scales, the Nasal Obstruction Symptom Evaluation Scale (NOSE), the 22-item Sinonasal Outcome Test (SNOT-22), and Likert scales. Objective outcomes included nasal peak inspiratory flow, nasal airway resistance, and minimum cross-sectional area. All outcomes were assessed preoperatively and 6 months postoperatively. The 36-item Optum SF-36v2 Health Survey mental component summary was used to assess mental well-being, with a score of less than 40 indicating poor mental well-being and a score 40 or higher indicating normal well-being. RESULTS: Mean (SD) patient age was 37.6 (12.9) years and 53 of 88 (60.2%) were women. The mental component summary defined impaired well-being in n = 24 (cases) and normal well-being in n = 64 (controls). There were improvements in the total study population across most nasal function outcomes and in both groups. After rhinoplasty, benefit was seen for both groups in visual analog scale (left side mean [SD] change, 18 [30]; P < .001 and right side mean [SD] change, 24 [30]; P < .001); NOSE (mean [SD] change, 1.35 [1.21]; P < .001); and SNOT-22 (mean [SD] change, 0.81 [0.88]; P < .001) scores. Nasal peak inspiratory flow improved for both groups (mean [SD] change, 32 [45] L/min; P < .001), while nasal airway resistance and minimum cross-sectional area remained similar (change in nasal airway resistance, 0.086 Pa/cm3/s; 95% CI, -0.007 Pa/cm3/s to 0.179 Pa/cm3/s and change in minimum cross-sectional area, -0.04 cm2; 95% CI, -0.21 cm2 to 0.13 cm2). Patients with poor mental health had similar improvements in nasal function compared with controls. CONCLUSIONS AND RELEVANCE: Rhinoplasty imparts similar benefits to nasal function assessed by patient-reported outcome measures and objective airflow measures regardless of preoperative mental health status. LEVEL OF EVIDENCE: 3.

Association of Mental Health Status With Perception of Nasal Function.

IMPORTANCE: Mental health issues are thought to be overrepresented among patients undergoing rhinoplasty and may be associated with patient presentation prior to surgery. OBJECTIVE: To assess the association of poor mental health with perception of nasal function. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study of patients presenting for airway assessment was performed from December 1, 2011, to October 31, 2015, at 2 tertiary rhinoplasty centers in Sydney, Australia. Mental health was independently defined preoperatively by the Mental Component Summary of the 36-item Short Form Health Survey version 2 (a score of <40 indicated poor mental well-being), the Rosenberg Self-Esteem Scale (a score of <15 indicated low self-esteem), and the Dysmorphic Concerns Questionnaire (a score of >11 indicated above-average dysmorphic concerns). MAIN OUTCOMES AND MEASURES: Nasal function was assessed with patient-reported outcome measures, including the Nasal Obstruction Symptom Evaluation Scale, the 22-item Sinonasal Outcome Test, a visual analog scale to rate ease of breathing on the left and right sides, and Likert scales to assess overall function and nasal obstruction. Nasal airflow was assessed by nasal peak inspiratory flow, nasal airway resistance, and minimum cross-sectional area. RESULTS: Among 495 patients in the study (302 women and 193 men; mean [SD] age, 36.5 [13.6] years), compared with patients with good mental health, those with poor mental health had poorer scores in all patient-reported outcome measures, including the visual analog scale for the left side (mean [SD], 51 [25] vs 42 [25]; P = .001), visual analog scale for the right side (mean [SD], 54 [24] vs 45 [26]; P < .001), Nasal Obstruction Symptom Evaluation Scale (mean [SD], 2.64 [0.95] vs 1.96 [1.04]; P < .001), 22-item Sinonasal Outcome Test (mean [SD], 2.14 [0.84] vs 1.33 [0.83]; P < .001), nasal obstruction (58 of 145 [40.2%] vs 83 of 350 [23.7%] with severe or worse obstruction; P < .001), and nasal function (72 of 145 [49.7%] vs 111 of 350 [31.8%] with poor or worse function; P < .001). Subclinical differences in nasal peak inspiratory flow could be demonstrated, but all other nasal airflow measures were similar. Low self-esteem produced a similar pattern, but dysmorphia did not. CONCLUSIONS AND RELEVANCE: Poor mental health status is associated with a poorer self-perception of nasal function compared with those who are mentally healthy with clinically similar nasal airflow. Clinicians should be aware that patients with poor mental health reporting obstructed airflow may in part be representing an extension of their negative emotions rather than true obstruction and may require further assessment prior to surgery. LEVEL OF EVIDENCE: NA.

Acute radiology rarely confirms sinus disease in suspected recurrent acute rhinosinusitis.

BACKGROUND: Episodic or recurrent sinonasal symptoms are often suspected as "sinus" in origin. With normal sinus radiology between events, the diagnosis of recurrent acute rhinosinusitis (RecARS) is made. However, other conditions can produce episodic symptoms. In this study we analyze acutely performed computed tomography (CT) in a population with suspected or self-diagnosed "sinus" disease. METHODS: Patients referred to a tertiary clinic for suspected RecARS were assessed. Sinus changes were defined by CT (initial assessment) and during the acute event, by a semiurgent CT performed during the symptomatic episode. Mucosal thickening, ostiomeatal compromise, and severe septal deformity were recorded. Symptom profile was assessed during both time-points with the 22-item Sino-Nasal Outcome Test (SNOT-22). RESULTS: Forty-eight patients (49.5 ± 14.7 years of age, 70.8% female) were assessed. At presentation, 75% were resolute in a diagnosis of "sinus." Baseline Lund-Mackay scores were <6 (median 0 [interquartile range 1]). Ostiomeatal compromise was 6.8% left and 4.5% right at baseline. Of the patients who returned for acute CT (n = 27), SNOT-22 and subdomains were similar to baseline. Septal deviation was similar (13.6% vs 15.3%). Acutely, ostiomeatal compromise was 0% left and 7.4% right (n = 2). Of these 2 patients with ostiomeatal compromise, 1 was diagnosed with RecARS (4%) and the other with triptan-responsive migraine, with incidental sinus changes. Final diagnosis was rhinitis (47%), headache/migraine (37%), and facial pain otherwise undefined (12.5%). CONCLUSION: Patients with a history of "recurrent acute sinusitis" and normal CT scans between episodes rarely have abnormal CT findings during acute exacerbations of symptoms. Antibiotics and surgical intervention are often inappropriate in this population.

Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

Allergic sensitization does not predispose to sinus inflammation in externalized paranasal sinuses.

BACKGROUND: Chronic rhinosinusitis (CRS) has a multifactorial etiology, with a debate about the role of inhalant allergy in the pathogenesis of CRS. OBJECTIVE: This study assessed the impact of allergy status on externalized paranasal sinuses after tumor resection to determine if a predisposition to inhalant allergy brought about additional inflammation after sinus surgery. METHODOLOGY: A case-control study was performed on patients who had no history of CRS who underwent paranasal sinus tumor resection. Allergic sensitization was defined by a positive serum ImmunoCAP test result. Outcomes were measured at least 6 months after surgery by using the modified Lund-MacKay endoscopic score and the 22-item Sino-Nasal Outcome test, with rhinitis, sleep, psychological, ear and/or facial, and sinus subscores to assess the impact of allergy status on mucosal inflammation. RESULTS: A total of 103 patients (53.44 ± 17.46 years; 46% women) were assessed. Of these, 61.17% were allergically sensitized at the time of surgery. Postsurgery endoscopic assessment was similar [the modified Lund-Mackay endoscopic score allergic sensitized 0.5 (1.7) versus nonallergic sensitized 0.0 (0.9); p = 0.15]. Sinonasal symptoms were also similar between the groups' 22-item Sino-Nasal Outcome test scores, allergic sensitized versus allergic nonsensitized, (allergic 28.9 ± 20.8 versus nonallergic 33.5 ± 19.7; p = 0.31), rhinitis score (5.9 ± 5.5 versus 6.4 ± 4.7; p = 0.66), sleep score (6.9 ± 5.9 versus 7.7 ± 4.8; p = 0.50), ear and/or facial symptom score (3.4 ± 3.6 versus 4.3 ± 3.3; p = 0.22), psychological score (6.9 ± 6.0 versus 8.3 ± 6.7; p = 0.29), and of nasal symptom score (6.4 ± 5.2 versus 7.0 ± 5.3; p = 0.61). CONCLUSIONS: Externalization of the sinuses in patients with inhalant allergy did not bring about significant additional inflammation in patients after tumor surgery.