Cytoskeleton (Hoboken, N.J.)最新文献

We describe a method for determining the ultrastructural organization of axons and varicosities of cultured dorsal root ganglion (DRG) neurons using cryogenic electron microscopy (cryo-EM). Cryo-EM reveals the dimensions, proximity, and overall organization of biological specimens in a near-native state, avoiding artifacts of fixation and heavy metal staining employed in classic thin section ultramicroscopy. Cryo-EM excels with thin specimens, and the axons of cryo-preserved cultured DRG neurons are the ideal thickness for high-resolution cryo-electron tomography. DRG neurons are a particularly interesting neuronal preparation because they can be isolated from animals of any age, providing a unique resource to examine age-related changes in axonal morphology. We provide a detailed, step-by-step protocol from DRG isolation and culturing, through cryo-preservation and data acquisition and analysis. We also provide a description for data processing in batch and how implementing deep-learning strategies can facilitate taking tilt-series data through the process of semi-automated tomographic segmentation required for quantitative descriptions of ultrastructural features. We use segmentations focused on the cytoskeletal elements of axons and varicosities of young and old cultured DRG neurons to highlight the approach.

Tubulin C-terminal tails undergo diverse post-translational modifications that regulate microtubule interactions with motors and severing enzymes. TTLL11, a member of the tubulin tyrosine ligase-like (TTLL) family, uniquely catalyzes glutamate addition to the terminal α-carboxyl group of both α- and β-primary tubulin tails. This linear C-terminal glutamylation enables the rescue of truncated tubulin variants and may support re-entry into the modification cycle. TTLL11 substrate specificity is determined by terminal residue identity rather than tubulin isotype. These findings expand the tubulin code and raise new questions about how linear and branched glutamylation are differentially recognized by microtubule-associated proteins.

Mitochondrial dysfunction and cytoskeletal disorganization are widely recognized hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Although these disorders differ in clinical presentation and etiology, accumulating evidence points to a shared cellular vulnerability at the intersection of mitochondrial dynamics and actin cytoskeletal regulation. In this review, we examine the emerging role of actin-mitochondria crosstalk as a convergent mechanism in neurodegeneration. We discuss how disruptions in actin filament remodeling, mitochondrial fission and fusion, organelle transport, and mitophagy contribute to neuronal dysfunction and loss across these diseases. Particular attention is given to disease-specific pathways, including cofilin-actin rod formation in AD, α-synuclein-driven actin disruption in PD, mutant huntingtin's effects on mitochondrial fragmentation in HD, and profilin-1-associated mitochondrial defects in ALS. By synthesizing findings from diverse models, we highlight how perturbations in the cytoskeleton-mitochondria interface may act as an upstream trigger and amplifier of neurodegenerative cascades. We also outline key knowledge gaps and propose future directions for research, with an emphasis on targeting actin-mitochondrial interactions as a potential therapeutic strategy across multiple neurodegenerative conditions.

Microtubules are noncovalent polymers assembled from α/β tubulin dimers. Their structure, dynamics and interaction with effectors are regulated through the expression of diverse tubulin isotypes and chemically diverse posttranslational modifications, also known as the "tubulin code." Understanding the biophysical correlates between tubulin sequence, posttranslational modifications, and microtubule structure and dynamics requires the ability to engineer tubulin and produce homogenous, chemically well-defined tubulin preparations. Here, we provide a protocol for the baculovirus expression and three-step purification of recombinant α1A/βIII tubulin in its tyrosinated, detyrosinated, Δ2 form as well as α-tailless form. Our protocol yields milligrams of pure, homogenous, and monodisperse recombinant human tubulin suitable for structural studies and in vitro reconstitution assays. Our system allows facile engineering of diverse tubulin variants, providing a necessary tool for understanding microtubule structure and dynamics, and the effects of the tubulin code on microtubule functions.

We developed LamelliQuant, an automated image analysis pipeline for quantifying lamellipodial protrusion and retraction dynamics from time-lapse microscopy of migrating cells. The workflow begins with the generation of kymographs along user-defined lines across lamellipodia, capturing edge motion over time. An image analysis algorithm extracts the cell edge trajectory and applies LOESS smoothing combined with peak detection to identify cycles of protrusion and retraction. LamelliQuant integrates ImageJ for image processing (movie import, ROI selection, kymograph generation) and R for quantitative analysis (smoothing and event detection), with user-adjustable parameters throughout to enable customization. We validated the pipeline by comparing automated measurements to manual tracking, demonstrating strong concordance and the ability to reproduce published results. LamelliQuant offers a robust, high-throughput alternative to manual kymograph analysis for investigating lamellipodia and membrane protrusion dynamics.

Long-distance intracellular transport of ionotropic glutamate receptors (iGluRs) is essential for proper excitatory synaptic function underlying learning and memory. Many neuropsychiatric and neurodegenerative conditions have abnormal iGluR transport and trafficking, leading to an intense interest in the mechanisms and factors regulating these processes. Although iGluRs and synaptic protein transport have been studied in cultured neurons, in vitro systems lack the specific connectivity of native circuits essential for the organization and regulation of compartmentalized synaptic signaling. Here, we describe an in vivo imaging approach that leverages the optical transparency of C. elegans to measure the transport of glutamate receptors in a fully intact neural system. Our workflow includes a standardized protocol for worm mounting, high-resolution imaging, and quantification of motor-driven iGluR transport in C. elegans. We discuss critical parameters for optimal signal-to-noise ratio, analysis, and reproducibility. Through years of optimization, we have established which fluorophores and genetic tools are the most effective and reproducible for in vivo transport imaging. These results provide a refined and reproducible framework for studying motor-driven iGluR transport in an intact nervous system and highlight important technical variables that can affect in vivo transport imaging.