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Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5β) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.

Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49-59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.

Background: Higher bone or metal glenoid offset in reverse shoulder arthroplasty (RSA) reduces scapular notching, improves range of motion (ROM), and reduces postoperative instability. This retrospective multicenter study compared two implant designs to evaluate the short-term clinical and radiologic results of bone increased offset RSA (BIO-RSA) and metal increased offset RSA (MIO-RSA) in reverse shoulder. We hypothesized no difference between groups., Methods: This study analyzed n = 62 BIO-RSA and n = 90 MIO-RSA cases with a mean follow-up of 29.7 ± 6.0 months (BIO-RSA, range 24-49 months) and 24.0 ± 1.1 months (MIO-RSA, range 22-28 months). A 145°-onlay humeral stem was utilized in BIO-RSA cases, while a 135°-semi-inlay humeral stem was implanted in all MIO-RSA cases. Preoperative and postoperative radiologic imaging was reviewed to identify signs of scapular notching. Additionally, lateralization was evaluated according to Erickson et al. The constant score, subjective shoulder value, and ROM were evaluated during the baseline and follow-up consultations, and the findings of both groups were subsequently compared., Results: Scapular notching was observed in 7.0% (n = 8) of MIO-RSA cases and 8.1% (n = 5) of BIO-RSA cases ( P  = .801). MIO-group had a higher lateralization angle ( P  = .020) and the BIO-group had a higher distalization angle ( P  = .005). At baseline, mean constant score in the MIO-RSA group was higher than in the BIO-RSA group ( P  < .001), and it significantly increased to 67.8 ± 12.1P (MIO-RSA) and 69.5 ± 12.3P (BIO-RSA) to a similar level ( P  = .399). ROM improved in both groups with no significant difference between the two groups at follow-up., Conclusion: BIO-RSA and MIO-RSA in two distinct implant designs provide comparable short-term outcomes with a similar increase in shoulder function with notable variations in the lateralization and distalization angles between both implants. Scapular notching was rarely seen and unaffected by the method of glenoid lateralization. Follow-up investigations of both techniques are necessary to complement and track changes in the long-term outcome., (© 2024 Published by Elsevier Inc. on behalf of American Shoulder and Elbow Surgeons.)

The Latarjet procedure is a proven and effective operation to treat anterior shoulder instability. Especially in cases with anterior glenoid bone loss, the Latarjet operation is the most popular procedure to restore glenoid anatomy and avoid further dislocations. Next to the re-creation of the missing glenoid bone, the sling effect of the conjoint tendon transferred between a split in the subscapularis muscle is an important "soft tissue stabilizer" of the humeral head. However, it has been shown that the inferior part of the subscapularis muscle tends to degenerate, leading to fatty infiltration of the muscle itself. Also, exposure through the subscapularis split is technically demanding, and there is a risk of nerve damage due to the pulling forces of the retractors during open surgery. When performing the procedure arthroscopically, extremely low and medial portals are necessary to find a correct angle for the glenoid drilling when approaching from anterior. Neurovascular structures may be at risk during these surgical steps. The aim of the flipped Latarjet procedure is to facilitate a safe and reliable arthroscopic operation to anteriorly stabilize the shoulder by transferring the coracoid to the deficient glenoid without splitting the subscapularis muscle while keeping the benefits of a sling effect of the conjoined tendon., Competing Interests: The authors report the following potential conflicts of interest or sources of funding: P.R. is a paid consultant for Arthrex. A.C. has a patent 11103292 issued to Arthrex. S.B. is employee of Arthrex. M.A. is employed at Arthrex GmbH. T.W. declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Full ICMJE author disclosure forms are available for this article online, as supplementary material., (© 2023 The Authors.)

Background: Short stems have become increasingly popular in reverse shoulder arthroplasty (RSA) due to their ability to preserve bone stock for revision surgery. However, short stems may be more at risk for malalignment or loosening, and commercially available stems have varied designs. The purpose of this study was to perform a multiplanar analysis of proximal humerus anatomy in patients with rotator cuff arthropathy to better define canal geometry and identify differences based on sex., Methods: A retrospective review was performed of a consecutive series of patients undergoing RSA for rotator cuff arthropathy. A total of 117 patients were identified with preoperative computed tomography scans. Measurements were undertaken following multiplanar reconstruction of the computed tomography scans. Measured parameters included the following: transition point (TP), anteroposterior (AP) and mediolateral (ML) distances, intramedullary (IM) and bone diameter, and cortical thickness. The TP was defined as the distance from the periosteal border of the greater tuberosity to the level of the IM canal where the endosteal borders became parallel. Measurements started at the metaphysis, and then proceeded 25 and 50 mm distal to the metaphysis followed by 10 mm increments thereafter. Each level was compared to the level above with t tests in the overall cohort and separately by sex. Height was correlated to ML-AP difference and IM diameter with Pearson correlation coefficient. Potential stem sizes that extended 50, 60, 70, and 80 mm from the metaphysis were analyzed to record the percentage of patients in whom the stem would reach past the TP., Results: The mean TP for all patients was 55.6 ± 7.4 mm (37.5-78.4) from the greater tuberosity, 53.3 ± 6.6 mm (37.5-67.0) in females and 58.1 ± 7.5 mm (41.9-78.4) in males. ML and AP distances and IM diameter became consistent at level 3 (mean, 83 mm distal to the greater tuberosity) in the overall cohort and in both sexes. Height positively correlated with IM diameter. Males had significantly larger IM diameters compared to females at all levels. Cortical thickness remained relatively consistent throughout the proximal humerus. A stem length of 70 mm would extend past the TP in 98% of patients., Conclusion: Humeral implants in RSA with a stem of at least 70 mm in length would extend distally past the TP in the majority of cases regardless of sex. At this point, the canal's area remains consistent which would facilitate diaphyseal fixation if required., (© 2023 The Author(s).)

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end-tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here, we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step, thereby enabling this approach in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance., Competing Interests: AD, RC, SR, Jv, TW No competing interests declared, (© 2023, Dema et al.)

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity., (© 2022 Stormo et al.)

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex. 1 , 2 In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures. 3 Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation, 4-6 the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells. 7 We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Many lysosome functions are determined by a lumenal pH of ∼5.0, including the activity of resident acid-activated hydrolases. Lysosome pH (pHlys) is often increased in neurodegenerative disorders and predicted to be decreased in cancers, making it a potential target for therapeutics to limit the progression of these diseases. Accurately measuring pHlys, however, is limited by currently used dyes that accumulate in multiple intracellular compartments and cannot be propagated in clonal cells for longitudinal studies or used for in vivo determinations. To resolve this limitation, we developed a genetically encoded ratiometric pHlys biosensor, pHLARE ( pH L ysosomal A ctivity RE porter), which localizes predominantly in lysosomes, has a dynamic range of pH 4.0 to 6.5, and can be stably expressed in cells. Using pHLARE we show decreased pHlys with inhibiting activity of the mammalian target of rapamycin complex 1 (mTORC1). Also, cancer cells from different tissue origins have a lower pHlys than untransformed cells, and stably expressing oncogenic RasV12 in untransformed cells is sufficient to decrease pHlys. pHLARE is a new tool to accurately measure pHlys for improved understanding of lysosome dynamics, which is increasingly considered a therapeutic target.

Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin 1,2 . However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn -/- microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn -/- microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration.

Background: The radiological phenomenon of subsidence following the implantation of uncemented short-stem reverse prostheses (USSP) has not yet been described. The purpose of this study was to describe the rate and potential risk factors for subsidence. We hypothesized that subsidence may be a frequent finding and that a subsidence of >5 mm (mm) is associated with an inferior clinical outcome., Methods: A total of 139 patients with an average age of 73 ± 9 years were included. The clinical and radiological outcome was evaluated at a minimum follow-up (FU) of 12 months., Results: No humeral component loosening was present at a mean FU of 18 (range, 12-51) months. Mean Constant Score (CS) and Subjective Shoulder Value (SSV) improved significantly from 34.3 ± 18.0 points and 37.0 ± 19.5% preoperatively to 72.2 ± 13.4 points and 80.3 ± 16.5% at final FU ( p < 0.001). The average subsidence of the USSP was 1.4 ± 3.7 mm. Subsidence of >5 mm was present in 15 patients (11%). No association between a subsidence >5 mm and CS or SSV was found ( p = 0.456, p = 0.527). However, a subsidence of >5 mm resulted in lower strength at final FU ( p = 0.022). Complications occurred in six cases (4.2%), and the revision rate was 3.5% (five cases)., Conclusions: Although subsidence of USSP is a frequent radiographic finding it is not associated with loosening of the component or a decrease in the clinical outcome at short term FU., Level of Evidence: Level 4, retrospective study.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions., (© 2019 Charafeddine et al.)

Adenosine triphosphate (ATP)-binding cassette subfamily A member 3 (ABCA3), a phospholipid transporter in lung lamellar bodies (LBs), is essential for the assembly of pulmonary surfactant and LB biogenesis. Mutations in the ABCA3 gene are an important genetic cause for respiratory distress syndrome in neonates and interstitial lung disease in children and adults, for which there is currently no cure. The aim of this study was to prove that disease causing misfolding ABCA3 mutations can be corrected in vitro and to investigate available options for correction. We stably expressed hemagglutinin (HA)-tagged wild-type ABCA3 or variants p.Q215K, p.M760R, p.A1046E, p.K1388N or p.G1421R in A549 cells and assessed correction by quantitation of ABCA3 processing products, their intracellular localization, resembling LB morphological integrity and analysis of functional transport activity. We showed that all mutant proteins except for M760R ABCA3 were rescued by the bithiazole correctors C13 and C17. These variants were also corrected by the chemical chaperone trimethylamine N-oxide and by low temperature. The identification of lead molecules C13 and C17 is an important step toward pharmacotherapy of ABCA3 misfolding-induced lung disease.