Symmetric cell division entails the equal distribution of cellular components to daughter cells. However, the mechanisms governing organelle segregation remain elusive. The endoplasmic reticulum (ER), comprising perinuclear sheets and peripheral tubules in interphase, serves as a central hub for sensing cellular states and coordinating other organelle dynamics. Here, we show that upon mitotic entry, the ER undergoes reverse redistribution: tubular ER accumulates around the centrosomes, while sheet-like ER relocates to the periphery. Mechanistically, the tubular ER protein Reticulon 4 (RTN4) is phosphorylated by cyclin-dependent kinase 1 (CDK1) during early mitosis. Phosphorylation promotes the interaction between RTN4 and Rab11, leading to the dynein-dependent enrichment of RTN4 around centrosomes and consequently driving the tubularization of the pericentrosomal ER. RTN4-mediated mitotic ER reorganization ensures symmetric distribution and inheritance of the ER, further contributing to the symmetric segregation of other organelles and mitotic fidelity. Thus, our study uncovers the mechanism of ER symmetry remodeling during early mitosis and its roles in organelle inheritance and mitotic progression.

Autophagy is a fundamental process for maintaining cell homeostasis, and STX17-mediated autophagosome-lysosome fusion is essential for cargo degradation and recycling. The translocation of STX17 to the autophagosome membrane is necessary for the fusion, yet the mechanism governing this process remains to be fully elucidated. Here, we show that following starvation, STX17 is acetylated at lysine 254 (K254), and this promotes the autophagosomal translocation of STX17 and subsequent autophagosome-lysosome fusion. Acetyltransferase GCN5 mediates STX17 K254 acetylation, which is counteracted by the deacetylase SIRT1. Moreover, autophagosomal translocation of K254-acetylated STX17 is mediated by myosin Ⅵ. Therefore, our research highlights the importance of acetylation and F-actin-based motor proteins in autophagosomal translocation of STX17 and autophagosome-lysosome fusion.

Amyloid precursor protein (APP), a type I transmembrane protein, is closely related to the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Aβ) is generated by sequential processing of APP in the Golgi apparatus and endosomes, and its toxicity leads to neuron dysfunction and neurodegeneration. APP is selectively shuttled between intracellular membrane compartments and ultimately transported into lysosomes. However, the mechanisms underlying APP sorting signals and lysosomal degradation are largely unclear. In this study, we show that the von Hippel‒Lindau (VHL) protein, a subunit of an E3 ligase, recognizes the cytoplasmic domain of APP and mediates its ubiquitination. VHL-mediated ubiquitination facilitates the sorting of membrane APP into intraluminal vesicles of multivesicular bodies (MVBs) and subsequent degradation in lysosomes. Therefore, the loss of VHL accelerates Aβ plaque deposition and memory deficits in AD model mice. Our findings reveal the role of VHL in restricting AD pathogenesis through ubiquitination-dependent MVB sorting and lysosomal degradation of APP.

Primary cilia are signaling hubs essential for development and homeostasis, and their formation is tightly regulated. Although F‑actin cytoskeleton is implicated in ciliogenesis, the role of branched F-actin networks remains controversial. Here we report that CCDC22 and LRCH2 antagonistically regulate ciliogenesis via different pools of branched F‑actin networks. CCDC22 stabilizes endosomal branched F-actin to maintain vesicle recycling; its loss promotes recycling endosomes toward centrosomes, thereby hyperactivating ciliogenesis. In vivo deletion of CCDC22 in mouse cerebellar granule cell precursors also increases ciliation, leading to motor defects. Whereas LRCH2, which binds to F‑actin and localizes to the centrosome as a CCDC22 interactor, sustains centrosomal branched F‑actin networks to promote ciliary vesicle recruitment and thereby facilitates ciliogenesis. Our findings reveal that endosomal and centrosomal branched F-actin provides a spatially restricted F-actin cytoskeletal pathway that regulates primary cilia formation.

β-Amyloid precursor protein (APP), an amyloid β-peptide (Aβ) generating protein, plays a central role in Alzheimer's disease (AD). Its intracellular trafficking and proteolytic processing are precisely regulated, but the mechanisms remain elusive. Here, we identified LAMTOR1 as a novel APP intracellular domain binding partner that sorts APP from early endosomes to late endosomes. Knockout of LAMTOR1 decreased APP trafficking to late endosomes, which promoted its amyloidogenic processing on early endosomes, resulting in increased Aβ. Conditional knockout of LAMTOR1 in Camk2a-expressing neurons impaired memory function in AD mouse model. Furthermore, the targeting of APP to late endosomes is mediated by adaptor protein complex 3 (AP-3). AP-3 bound to LAMTOR1, and its depletion caused accumulation of LAMTOR1 and APP within early endosomes, subsequently increasing Aβ. Thus, LAMTOR1 functions as a regulator of APP trafficking to late endosomes and may serve as a potential therapeutic target for AD.

The primary cilium serves as a crucial signaling hub that integrates extracellular cues to coordinate development and maintain homeostasis, and its formation (ciliogenesis) is strictly regulated. Given the complex and spatially distinct organization of the actin filament cytoskeleton, the precise contributions of branched F-actin to ciliogenesis remain poorly understood. Here, we show that centrosomal branched actin filaments (c-BFA) facilitate ciliogenesis by directing the ARP2/3 nucleation‑promoting factor VCA to the centrosome. We identify LRCH2 as an actin‑binding protein that stabilizes c-BFA, thereby promoting ciliary vesicle docking at the mother centriole and enhancing ciliogenesis. In contrast, CCDC22, a known early endosome‑localized protein, regulates endosomal vesicle sorting by stabilizing non‑centrosomal branched actin filaments (nc-BFA), which consequently suppresses ciliary vesicle recruitment to the centrosome and inhibits ciliogenesis. Collectively, LRCH2 and CCDC22 antagonistically regulate branched actin networks at the centrosome and in the cytoplasm, thereby maintaining ciliogenesis homeostasis. Conditional knockout of Ccdc22 in mouse cerebellar granule cell precursors results in an increased proportion of ciliated cells and impairs balance function. Our findings highlight the distinct and opposing roles of c-BFA and nc-BFA in primary ciliogenesis.

Desmosomes are cell-cell adhesive junctions that provide structural integrity and mechanical resistance to tissues. Disruptions in desmosome organization lead to severe cardiac and dermatological disorders. Emerging evidence reveals that desmosomes exhibit dynamic behaviors during remodeling. However, the mechanisms that initiate and regulate these dynamics under physiological conditions, and their correlation with desmosome-related disorders remain elusive. Here, we uncover the role of coiled-coil domain-containing 120 (CCDC120) in linking desmosomal organization to cardiac function via liquid-liquid phase separation (LLPS). CCDC120 localizes to desmosomes and is required for desmosomal integrity. CCDC120 exhibits LLPS properties and co-condenses with the desmosomal component plakophilin-2 (PKP2) during desmosomal assembly, forming dynamic condensates crucial for preserving desmosomal structure and junction stability. Moreover, CCDC120 condensates are modulated by PKCα phosphorylation. Loss of CCDC120 or altered CCDC120 phase separation properties leads to intercalated disc structure impairment and cardiac dysfunction in mice. Our findings propose a model wherein CCDC120 phase separation orchestrates desmosomal integrity, thereby establishing connections between desmosomal dynamics and the molecular etiology of cardiac dysfunction.

Mitochondrial transport and distribution are crucial for cellular homeostasis, yet whether and how they are regulated by endoplasmic reticulum (ER)-mitochondria contact sites remains unclear. Here, we demonstrate that the ER protein atlastin-2 (ATL2) orchestrates mitochondrial transport and distribution by promoting assembly of the transport machinery at ER-mitochondria contact sites. Mechanistically, ATL2 recruits the adaptor trafficking kinesin-binding protein 1 (TRAK1) to the ER membrane, strengthening the interaction of TRAK1 with the mitochondrial transport adaptor MIRO1 to promote anterograde mitochondrial transport. Loss of ATL2 disrupts this process, leading to perinuclear mitochondrial clustering. We further find that ATL2 stabilizes ER-mitochondria contact sites by interacting with MFN2, providing a platform for mitochondrial transport complex assembly. Moreover, in hypoxia, ATL2 is ubiquitinated at lysine 567 by the E3 ligase SYVN1, leading to its degradation and a resulting defect in mitochondrial distribution. Our findings elucidate a novel ER-mediated mechanism for mitochondrial transport.

Genomic integrity in mammalian cells relies on faithful chromosome segregation. Kinetochores capture microtubules and establish robust kinetochore-microtubule attachment to achieve accurate segregation. The MIS12 complex, a tetramer comprising MIS12, DSN1, NSL1, and PMF1, plays a critical role in kinetochore-microtubule attachment. However, how the MIS12 complex functions at the kinetochore-microtubule interface is not fully understood. Here, we found that MIS12 is phosphorylated at Ser177 by NEK2A from prophase to prometaphase. Phosphorylation of MIS12 expands the projection of the outer kinetochore, known as the fibrous corona, thus facilitating the attachment of kinetochores to microtubules. When chromosomes align at the equatorial plate, Ser177 on MIS12 is dephosphorylated by PP1, which is required for kinetochore compaction and end-on attachment conversion. We uncovered that the dynamic phosphorylation of MIS12 regulates the expansion-compaction transition of the outermost layer of kinetochores, ensuring accurate kinetochore-microtubule attachment and faithful chromosome segregation.

Planar cell polarity (PCP) is an evolutionarily conserved process for development and morphogenesis in metazoans. The well-organized polarity pattern in cells is established by the asymmetric distribution of two core protein complexes on opposite sides of the cell membrane. The Van Gogh-like (VANGL)-PRICKLE (PK) pair is one of these two key regulators; however, their structural information and detailed functions have been unclear. Here, we present five cryo-electron microscopy structures of human VANGL1, VANGL2, and their complexes with PK1 at resolutions of 2.2–3.0 Å. Through biochemical and cell imaging experiments, we decipher the molecular details of the VANGL-PK interaction. Furthermore, we reveal that PK1 can target VANGL-containing intracellular vesicles to the peripheral cell membrane. These findings provide a solid foundation to understand the explicit interaction between VANGL and PK while opening new avenues for subsequent studies of the PCP pathway. The authors present cryo-electron structures of human VANGL1, VANGL2 and their complexes with PK1, unveiling the molecular underpinnings of the VANGL-PK interaction and paving the way for future investigations into the planar cell polarity pathway

The spindle assembly checkpoint (SAC) ensures chromosome segregation fidelity by manipulating unattached kinetochore-dependent assembly of the mitotic checkpoint complex (MCC). The MCC binds to and inhibits the anaphase promoting complex/cyclosome (APC/C) to postpone mitotic exit. However, the mechanism by which unattached kinetochores mediate MCC formation is not yet fully understood. Here, it is shown that CCDC68 is an outer kinetochore protein that preferentially localizes to unattached kinetochores. Furthermore, CCDC68 interacts with the SAC factor CDC20 to inhibit its autoubiquitination and MCC disassembly. Therefore, CCDC68 restrains APC/C activation to ensure a robust SAC and allow sufficient time for chromosome alignment, thus ensuring chromosomal stability. Hence, the study reveals that CCDC68 is required for CDC20-dependent MCC stabilization to maintain mitotic checkpoint activation.

In animal cells, the dysregulation of centrosome duplication and cohesion maintenance leads to abnormal spindle assembly and chromosomal instability, contributing to developmental disorders and tumorigenesis. However, the molecular mechanisms involved in maintaining accurate centrosome number control and tethering are not fully understood. Here, we identified coiled -coil domain -containing 102A (CCDC102A) as a centrosomal protein exhibiting a barrel -like structure in the proximal regions of parent centrioles, where it prevents centrosome overduplication by restricting interactions between Cep192 and Cep152 on centrosomes, thereby ensuring bipolar spindle formation. Additionally, CCDC102A regulates the centrosome linker by recruiting and binding C-Nap1; it is removed from the centrosome after Nek2A-mediated phosphorylation at the onset of mitosis. Overall, our results indicate that CCDC102A participates in controlling centrosome number and maintaining centrosome cohesion, suggesting that a well -tuned system regulates centrosome structure and function throughout the cell cycle.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

Autophagy is a fundamental process for maintaining cell homeostasis, and STX17-mediated autophagosome-lysosome fusion is essential for cargo degradation and recycling. The translocation of STX17 to the autophagosomal membrane is necessary for the fusion, yet the mechanism governing this process remains largely unknown. Here, we show that the cytosolic, but not membrane-inserted, STX17 translocates onto autophagosomes during autophagy. Following starvation, STX17 is acetylated at lysine 254 (K254) which is accessible only in cytosolic STX17, and this promotes the autophagosomal translocation of STX17 and subsequent autophagosome-lysosome fusion. The cytosolic acetyltransferase GCN5 mediates STX17 K254 acetylation, which is counteracted by the deacetylase SIRT1. Moreover, autophagosomal translocation of K254-acetylated STX17 is mediated by myosin Ⅵ. Therefore, our research proposes the cytosolic source of autophagosomal STX17 and highlights the importance of acetylation and F-actin-based motor proteins in autophagosome-lysosome fusion.

Autophagy and Hippo signalling pathways both play important roles in cell homeostasis and are often involved in tumourigenesis. However, the crosstalk between these two signal pathways in response to stress conditions, such as nutrient deficiency, is incompletely understood. Here, we show that vesicular localised coiled-coil domain containing 115 (CCDC115) inhibits autophagy as well as Hippo signalling pathway under starvation. Moreover, we show that CCDC115 interacts with the HOPS complex. This interaction competes with STX17, thus inhibiting the fusion of autophagosomes with lysosomes. Hence, CCDC115 inhibits the autophagic degradation of yes-associated protein (YAP), thereby promoting cell proliferation in nutrient-restricted situation.

Centrosomes are composed of centrioles surrounded by pericentriolar material. The two centrioles in G1 phase are distinguished by the localization of their appendages in the distal and subdistal regions; the centriole possessing both types of appendage is older and referred to as the mother centriole, whereas the other centriole lacking appendages is the daughter centriole. Both distal and subdistal appendages in vertebrate cells consist of multiple proteins assembled in a hierarchical manner. Distal appendages function mainly in the initial process of ciliogenesis, and subdistal appendages are involved in microtubule anchoring, mitotic spindle regulation and maintenance of ciliary signaling. Mutations in genes encoding components of both appendage types are implicated in ciliopathies and developmental defects. In this Review, we discuss recent advances in knowledge regarding the composition and assembly of centriolar appendages, as well as their roles in development and disease.

Centrosomes are composed of centrioles surrounded by pericentriolar material. The two centrioles in G1 phase are distinguished by the localization of their appendages in the distal and subdistal regions; the centriole possessing both types of appendage is older and referred to as the mother centriole, whereas the other centriole lacking appendages is the daughter centriole. Both distal and subdistal appendages in vertebrate cells consist of multiple proteins assembled in a hierarchical manner. Distal appendages function mainly in the initial process of ciliogenesis, and subdistal appendages are involved in microtubule anchoring, mitotic spindle regulation and maintenance of ciliary signaling. Mutations in genes encoding components of both appendage types are implicated in ciliopathies and developmental defects. In this Review, we discuss recent advances in knowledge regarding the composition and assembly of centriolar appendages, as well as their roles in development and disease.

ABSTRACT Proper microtubule dynamics are critical for neuronal morphogenesis and functions, and their dysregulation results in neurological disorders and regeneration failure. Superior cervical ganglion-10 (SCG10, also known as stathmin-2 or STMN2) is a well-known regulator of microtubule dynamics in neurons, but its functions in the peripheral nervous system remain largely unknown. Here, we show that Scg10 knockout mice exhibit severely progressive motor and sensory dysfunctions with significant sciatic nerve myelination deficits and neuromuscular degeneration. Additionally, increased microtubule stability, shown by a significant increase in tubulin acetylation and decrease in tubulin tyrosination, and decreased axonal transport were observed in Scg10 knockout dorsal root ganglion (DRG) neurons. Furthermore, SCG10 depletion impaired axon regeneration in both injured mouse sciatic nerve and cultured DRG neurons following replating, and the impaired axon regeneration was found to be induced by a lack of SCG10-mediated microtubule dynamics in the neurons. Thus, our results highlight the importance of SCG10 in peripheral axon maintenance and regeneration.

Extracting the position of individual molecular probes with high precision is the basis and core of super-resolution microscopy. However, with the expectation of low-light conditions in life science research, the signal-to-noise ratio (SNR) decreases and signal extraction faces a great challenge. Here, based on temporally modulating the fluorescence emission at certain periodical patterns, we achieved super-resolution imaging with high sensitivity by largely suppressing the background noise. We propose simple bright-dim (BD) fluorescent modulation and delicate control by phase-modulated excitation. We demonstrate that the strategy can effectively enhance signal extraction in both sparsely and densely labeled biological samples, and thus improve the efficiency and precision of super-resolution imaging. This active modulation technique is generally applicable to various fluorescent labels, super-resolution techniques, and advanced algorithms, allowing a wide range of bioimaging applications.

Single-molecule localization microscopy (SMLM) has been widely used in biological imaging due to its ultrahigh spatial resolution. However, due to the strategy of reducing photodamage to living cells, the fluorescence signals of emitters are usually weak and the detector noises become non-negligible, which leads to localization misalignments and signal losses, thus deteriorating the imaging capability of SMLM. Here, we propose an active modulation method to control the fluorescence of the probe emitters. It actually marks the emitters with artificial blinking character, which directly distinguishes weak signals from multiple detector noises. We demonstrated from simulations and experiments that this method improves the signal-to-noise ratio by about 10 dB over the non-modulated method and boosts the sensitivity of single-molecule localization down to -4 dB, which significantly reduces localization misalignments and signal losses in SMLM. This signal-noise decoupling strategy is generally applicable to the super-resolution system with versatile labeled probes to improve their imaging capability. We also showed its application to the densely labeled sample, showing its flexibility in super-resolution nanoscopy.