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Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in tumorigenesis, little is known about the roles of mitochondrial dynamics in metastasis, the major cause of cancer death. In the present study, we found a marked upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1) expression in human invasive breast carcinoma and metastases to lymph nodes. Compared with non-metastatic breast cancer cells, mitochondria also were more fragmented in metastatic breast cancer cells that express higher levels of total and active Drp1 and less mitochondrial fusion protein 1 (Mfn1). Silencing Drp1 or overexpression of Mfn1 resulted in mitochondria elongation or clusters, respectively, and significantly suppressed metastatic abilities of breast cancer cells. In contrast, silencing Mfn proteins led to mitochondrial fragmentation and enhanced metastatic abilities of breast cancer cells. Interestingly, these manipulations of mitochondrial dynamics altered the subcellular distribution of mitochondria in breast cancer cells. For example, silencing Drp1 or overexpression of Mfn1 inhibited lamellipodia formation, a key step for cancer metastasis, and suppressed chemoattractant-induced recruitment of mitochondria to lamellipodial regions. Conversely, silencing Mfn proteins resulted in more cell spreading and lamellipodia formation, causing accumulation of more mitochondria in lamellipodia regions. More importantly, treatment with a mitochondrial uncoupling agent or adenosine triphosphate synthesis inhibitor reduced lamellipodia formation and decreased breast cancer cell migration and invasion, suggesting a functional importance of mitochondria in breast cancer metastasis. Together, our findings show a new role and mechanism for regulation of cancer cell migration and invasion by mitochondrial dynamics. Thus targeting dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing breast cancer metastasis.

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7⁻/⁻ erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119⁺/CD71High stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.

Key Points The γ-tubulin small complex (γTuSC) alone can assemble into ring complexes with microtubule-like symmetry. The structures of γ-tubulin complexes suggest that they serve as microtubule templates. The γ-tubulin complex proteins (GCPs) are conserved in sequence, overall structure and their ability to bind γ-tubulin. The conformation of the γTuSC may play a part in regulating its microtubule-nucleating activity. A revised model of γ-tubulin ring complex (γTuRC) assembly, in which all of the GCPs are incorporated directly into the ring, has been proposed. The attachment of the γTuRC to both centrosomal and non-centrosomal sites is linked to its activation. Microtubule nucleation is regulated by the γ-tubulin small complex (γTuSC) and the γ-tubulin ring complex (γTuRC). Recent structural work, including the crystallographic analysis of γ-tubulin complex protein 4 (GCP4), provides new insights into the mechanism of γTuRC-based microtubule nucleation, confirming the hypothesis that the γTuRC functions as a microtubule template. Microtubule nucleation is regulated by the γ-tubulin ring complex (γTuRC) and related γ-tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that the γTuRC functions as a microtubule template. The first crystallographic analysis of a non-γ-tubulin γTuRC component (γ-tubulin complex protein 4 (GCP4)) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between its attachment at specific cellular sites and its activation.

Significance The normal function of the Huntingtin (HTT) protein is emerging. Here we report that selective autophagy requires an intact HTT protein in Drosophila and mouse CNS. We describe similarities in structure and binding activity between the C-terminal domain of HTT and the yeast autophagy scaffold protein Atg11, suggesting that HTT may normally function as a scaffold for various types of selective autophagy. Mice expressing an expanded repeat form of HTT also show deficits in protein clearance. Because autophagy is critical for clearance of cellular proteins, including mutant HTT, the impairment of normal HTT function by the polyQ expansion could suppress activity of the autophagy machinery. These results may have important implications when evaluating therapeutic strategies for HD. Although dominant gain-of-function triplet repeat expansions in the Huntingtin ( HTT ) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51–like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.

The evolutionarily conserved MAP65 family proteins bundle anti-parallel microtubules (MTs). In Arabidopsis thaliana, mutations in the MAP65-3 gene lead to serious defects in MT organization in the phragmoplast and cause failures in cytokinesis. However, the functions of other Arabidopsis MAP65 isoforms are largely unknown. MAP65 functions were analyzed based on genetic interactions among different map65 mutations. Live-cell imaging and immunolocalization experiments revealed dynamic activities of two closely related MAP65 proteins in dividing cells. The map65-4 mutation caused synthetic lethality with map65-3 although map65-4 alone did not cause a noticeable phenotype. Furthermore, the introduction of an extra copy of the MAP65-4 gene significantly suppressed defects in cytokinesis and seedling growth caused by map65-3 because of restoring MT engagement in the spindle midzone. During mitosis, MAP65-4 first appeared at the preprophase band and persisted at the cortical division site afterwards. It was also concentrated on MTs in the spindle midzone and the phragmoplast. In the absence of MAP65-3, MAP65-4 exhibited greatly enhanced localization in the midzone of developing phragmoplast. Therefore, we have uncovered redundant but differential contributions of MAP65-3 and MAP65-4 to engaging and bundling anti-parallel MTs in the phragmoplast and disclosed a novel action of MAP65-4 at the cortical cell division site.

The significance of autophagy for signal transduction has been unclear. Autophagy negatively regulates Wnt signalling by promoting Dishevelled (Dvl) degradation. The von Hippel-Lindau protein ubiquitylates Dvl to faciliatate its recruitment to autophagosomes through p62. In eukaryotic cells, autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses. Autophagy is regulated by cell signalling such as the mTOR (mammalian target of rapamycin) pathway. However, the significance of autophagy in modulation of signal transduction is unclear. Here we show that autophagy negatively regulates Wnt signalling by promoting Dishevelled (Dvl) degradation. Von Hippel–Lindau protein-mediated ubiquitylation is critical for the binding of Dvl2 to p62, which in turn facilitates the aggregation and the LC3-mediated autophagosome recruitment of Dvl2 under starvation; the ubiquitylated Dvl2 aggregates are ultimately degraded through the autophagy–lysosome pathway. Moreover, a reverse correlation between Dvl expression and autophagy is observed in late stages of colon cancer development, indicating that autophagy may contribute to the aberrant activation of Wnt signalling in tumour formation.

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5 , essential for the formation of autophagosomes, markedly impairs the progression of KRas G12D -driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5- mutant KRas G12D -driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas G12D -driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity. Autophagy prolongs the survival of cells in stressful conditions but its role in cancer is unclear. Here, Rao et al . show that loss of the autophagic protein Atg5 enhanced cancer incidence but impaired tumour progression in a mouse model of lung cancer.

During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein. Hearts from these mice had enhanced autophagy, but normal morphology and function. We crossed these mice with CryABR120G mice, a model of DRC in which autophagy is significantly attenuated in the heart, to test the functional significance of autophagy activation in a proteotoxic model of heart failure. Sustained Atg7-induced autophagy in the CryABR120G hearts decreased interstitial fibrosis, ameliorated ventricular dysfunction, decreased cardiac hypertrophy, reduced intracellular aggregates and prolonged survival. To determine whether different methods of autophagy upregulation have additive or even synergistic benefits, we subjected the autophagy-deficient CryABR120G mice and the Atg7-crossed CryABR120G mice to voluntary exercise, which also upregulates autophagy. The entire exercised Atg7-crossed CryABR120G cohort survived to 7 months. These findings suggest that activating autophagy may be a viable therapeutic strategy for improving cardiac performance under proteotoxic conditions.