Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of ...actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ∼180 to 190 nanometers. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin—based cytoskeleton.
Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on ...mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.
Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, ...we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites.
Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene ...delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.
Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified ...photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.
Chronic inflammation plays a central role in hepatocellular carcinoma (HCC), but the contribution of hepatocytes to tumor-associated inflammation is not clear. Here, we report that the zinc finger ...transcription factor Miz1 restricted hepatocyte-driven inflammation to suppress HCC, independently of its transcriptional activity. Miz1 was downregulated in HCC mouse models and a substantial fraction of HCC patients. Hepatocyte-specific Miz1 deletion in mice generated a distinct sub-group of hepatocytes that produced pro-inflammatory cytokines and chemokines, which skewed the polarization of the tumor-infiltrating macrophages toward pro-inflammatory phenotypes to promote HCC. Mechanistically, Miz1 sequestrated the oncoprotein metadherin (MTDH), preventing MTDH from promoting transcription factor nuclear factor κB (NF-κB) activation. A distinct sub-group of pro-inflammatory cytokine-producing hepatocytes was also seen in a subset of HCC patients. In addition, Miz1 expression inversely correated with disease recurrence and poor prognosis in HCC patients. Our findings identify Miz1 as a tumor suppressor that prevents hepatocytes from driving inflammation in HCC.
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•Miz1 suppresses liver cancer independently of its transcriptional activity•Miz1 restricts the ability of hepatocytes to drive macrophage-dependent inflammation•Miz1 prevents oncoprotein MTDH from promoting hepatocyte NF-κB activity•Miz1 expression inversely correlates with recurrence and poor prognosis in HCC patients
Chronic inflammation plays a crucial role in hepatocellular carcinoma (HCC), but the contribution of tumor hepatocytes to tumor-associated inflammation remains unclear. Zhang et al. find that loss of the transcription factor Miz1 in hepatocytes promotes NF-κB activation, producing a distinct sub-cluster of tumor hepatocytes that skew tumor-infiltrating macrophages toward a pro-inflammatory phenotype and drive inflammation in HCC.
With the rapid development of the advanced manufacturing industry, equipment requirements are becoming increasingly stringent. Since metallic materials often present failure problems resulting from ...wear due to extreme service conditions, researchers have developed various methods to improve their properties. Laser shock peening (LSP) is a highly efficacious mechanical surface modification technique utilized to enhance the microstructure of the near-surface layer of metallic materials, which improves mechanical properties such as wear resistance and solves failure problems. In this work, we summarize the fundamental principles of LSP and laser-induced plasma shock waves, along with the development of this technique. In addition, exemplary cases of LSP treatment used for wear resistance improvement in metallic materials of various nature, including conventional metallic materials, laser additively manufactured parts, and laser cladding coatings, are outlined in detail. We further discuss the mechanism by which the microhardness enhancement, grain refinement, and beneficial residual stress are imparted to metallic materials by using LSP treatment, resulting in a significant improvement in wear resistance. This work serves as an important reference for researchers to further explore the fundamentals and the metallic material wear resistance enhancement mechanism of LSP.
G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB
...), we discovered periodically repeating clusters of CB
hotspots within the axons of neurons. We observed these CB
hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB
signaling. Furthermore, we found that CB
hotspot periodicity increased upon CB
agonist application, and these activated CB
displayed less dynamic movement compared to non-activated CB
. Our results suggest that CB
forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.
The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been ...linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors.
Many animals are capable of changing gait with speed of locomotion. The neural basis of gait control and its dependence on speed are not fully understood. Mice normally use a single "trotting" gait ...while running at all speeds, either over ground or on a treadmill. Transgenic mouse mutants in which the trotting is replaced by hopping also lack a speed-dependent change in gait. Here we describe a transgenic mouse model in which the V2a interneurons have been ablated by targeted expression of diphtheria toxin A chain (DTA) under the control of the Chx10 gene promoter (Chx10::DTA mice). Chx10::DTA mice show normal trotting gait at slow speeds but transition to a galloping gait as speed increases. Although left-right limb coordination is altered in Chx10::DTA mice at fast speed, alternation of forelegs and hindlegs and the relative duration of swing and stance phases for individual limbs is unchanged compared with wild-type mice. The speed-dependent loss of left-right alternation is recapitulated during drug-induced fictive locomotion in spinal cords isolated from neonatal Chx10::DTA mice, and high-speed fictive locomotion evoked by caudal spinal cord stimulation also shows synchronous left-right bursting. These results show that spinal V2a interneurons are required for maintaining left-right alternation at high speeds. Whether animals that generate galloping or hopping gaits, characterized by synchronous movement of left and right forelegs and hindlegs, have lost or modified the function of V2a interneurons is an intriguing question.
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