Endosomal microautophagy is an integrated part of the autophagic response to amino acid starvation.

Starvation is a fundamental type of stress naturally occurring in biological systems. All organisms have therefore evolved different safeguard mechanisms to cope with deficiencies in various types of nutrients. Cells, from yeast to humans, typically respond to amino acid starvation by initiating degradation of cellular components by inducing autophagy. This degradation releases metabolic building blocks to sustain essential core cellular processes. Increasing evidence indicates that starvation-induced autophagy also acts to prepare cells for prolonged starvation by degrading key regulators of different cellular processes. In a recent study, we found that within the first hours of amino acid starvation cells elicit an autophagic response causing rapid degradation of specific proteins. The response is executed independently of both MTOR and canonical macroautophagy. Based on RNAi-mediated knockdown of essential components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery and electron microscopy we conclude that the response relies on some sort of endosomal microautophagy, hence vesicle budding into endosomes. Substantiated by the different substrates that are selectively degraded by this novel pathway we propose that the response predominantly acts to prepare cells for prolonged starvation. Intriguingly, this includes shutting down selective macroautophagy in preparation for a massive induction of bulk macroautophagy.

Starvation induces rapid degradation of selective autophagy receptors by endosomal microautophagy.

It is not clear to what extent starvation-induced autophagy affects the proteome on a global scale and whether it is selective. In this study, we report based on quantitative proteomics that cells during the first 4 h of acute starvation elicit lysosomal degradation of up to 2-3% of the proteome. The most significant changes are caused by an immediate autophagic response elicited by shortage of amino acids but executed independently of mechanistic target of rapamycin and macroautophagy. Intriguingly, the autophagy receptors p62/SQSTM1, NBR1, TAX1BP1, NDP52, and NCOA4 are among the most efficiently degraded substrates. Already 1 h after induction of starvation, they are rapidly degraded by a process that selectively delivers autophagy receptors to vesicles inside late endosomes/multivesicular bodies depending on the endosomal sorting complex required for transport III (ESCRT-III). Our data support a model in which amino acid deprivation elicits endocytosis of specific membrane receptors, induction of macroautophagy, and rapid degradation of autophagy receptors by endosomal microautophagy.

Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication.

During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation has an impact on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites, and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phospho-mimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signalling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components.

To maintain genome function and stability, DNA sequence and its organization into chromatin must be duplicated during cell division. Understanding how entire chromosomes are copied remains a major challenge. Here, we use nascent chromatin capture (NCC) to profile chromatin proteome dynamics during replication in human cells. NCC relies on biotin-dUTP labelling of replicating DNA, affinity purification and quantitative proteomics. Comparing nascent chromatin with mature post-replicative chromatin, we provide association dynamics for 3,995 proteins. The replication machinery and 485 chromatin factors such as CAF-1, DNMT1 and SUV39h1 are enriched in nascent chromatin, whereas 170 factors including histone H1, DNMT3, MBD1-3 and PRC1 show delayed association. This correlates with H4K5K12diAc removal and H3K9me1 accumulation, whereas H3K27me3 and H3K9me3 remain unchanged. Finally, we combine NCC enrichment with experimentally derived chromatin probabilities to predict a function in nascent chromatin for 93 uncharacterized proteins, and identify FAM111A as a replication factor required for PCNA loading. Together, this provides an extensive resource to understand genome and epigenome maintenance.

New histone supply regulates replication fork speed and PCNA unloading.

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.

Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption.

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage.

Lapatinib, a dual inhibitor of ErbB-1/-2 receptors, enhances effects of combination chemotherapy in bladder cancer cells.

Survival rate of patients diagnosed with the invasive form of bladder cancer is low suggesting an urgent need to implement novel treatments. GTC (gemcitabine, paclitaxel and cisplatin) is a new chemotherapeutic regimen, which has shown promise in clinical trials. Given that receptor tyrosine kinases of the ErbB family are overexpressed in a high proportion of metastatic bladder tumours, approaches involving small-molecule inhibitors of ErbB receptors in combination with conventional cytostatic drugs are of potential interest. Here, we show that the dual inhibitor of ErbB receptors, lapatinib, enhances cytostatic and induces cytotoxic effects of GTC in two bladder cancer cell lines which differ with regard to expression levels of proteins taking part in the ErbB pathway. Lapatinib inhibited phosphorylation of ErbB receptors and also reduced the level of phosphorylated AKT. Flow cytometry analysis demonstrated that GTC treatment affects cell cycle distribution differently in the presence or absence of lapatinib. In RT112 cells, which express high levels of ErbB receptors and harbour wild-type p53, combined GTC/lapatinib treatment resulted in the phosphorylation of p53 at Ser46 and accumulation of sub-G1 cell populations. Our data indicate that a combinatorial approach involving GTC and lapatinib may have therapeutic potential in a subset of bladder tumours depending on the genetic context.

Direct repression of cyclin D1 by SIP1 attenuates cell cycle progression in cells undergoing an epithelial mesenchymal transition.

Zinc finger transcription factors of the Snail/Slug and ZEB-1/SIP1 families control epithelial-mesenchymal transitions in development in cancer. Here, we studied SIP1-regulated mesenchymal conversion of epidermoid A431 cells. We found that concomitant with inducing invasive phenotype, SIP1 inhibited expression of cyclin D1 and induced hypophosphorylation of the Rb tumor suppressor protein. Repression of cyclin D1 was caused by direct binding of SIP1 to three sequence elements in the cyclin D1 gene promoter. By expressing exogenous cyclin D1 in A431/SIP1 cells and using RNA interference, we demonstrated that the repression of cyclin D1 gene by SIP1 was necessary and sufficient for Rb hypophosphorylation and accumulation of cells in G1 phase. A431 cells expressing SIP1 along with exogenous cyclin D1 were highly invasive, indicating that SIP1-regulated invasion is independent of attenuation of G1/S progression. However, in another epithelial-mesenchymal transition model, gradual mesenchymal conversion of A431 cells induced by a dominant negative mutant of E-cadherin produced no effect on the cell cycle. We suggest that impaired G1/S phase progression is a general feature of cells that have undergone EMT induced by transcription factors of the Snail/Slug and ZEB-1/SIP1 families.

Characterization of E-cadherin-dependent and -independent events in a new model of c-Fos-mediated epithelial-mesenchymal transition.

Fos proteins have been implicated in control of tumorigenesis-related genetic programs including invasion, angiogenesis, cell proliferation and apoptosis. In this study, we demonstrate that c-Fos is able to induce mesenchymal transition in murine tumorigenic epithelial cell lines. Expression of c-Fos in MT1TC1 cells led to prominent alterations in cell morphology, increased expression of mesenchymal markers, vimentin and S100A4, DNA methylation-dependent down-regulation of E-cadherin and abrogation of cell-cell adhesion. In addition, c-Fos induced a strong beta-catenin-independent proliferative response in MT1TC1 cells and stimulated cell motility, invasion and adhesion to different extracellular matrix proteins. To explore whether loss of E-cadherin plays a role in c-Fos-mediated mesenchymal transition, we expressed wild-type E-cadherin and two different E-cadherin mutants in MT1TC1/c-fos cells. Expression of wild-type E-cadherin restored epithelioid morphology and enhanced cellular levels of catenins. However, exogenous E-cadherin did not influence expression of c-Fos-dependent genes, only partly suppressed growth of MT1TC1/c-fos cells and produced no effect on c-Fos-stimulated cell motility and invasion in matrigel. On the other hand, re-expression of E-cadherin specifically negated c-Fos-induced adhesion to collagen type I, but not to laminin or fibronectin. Of interest, mutant E-cadherin which lacks the ability to form functional adhesive complexes had an opposite, potentiating effect on cell adhesion to collagen I. These data suggest that cell adhesion to collagen I is regulated by the functional state of E-cadherin. Overall, our data demonstrate that, with the exception of adhesion to collagen I, c-Fos is dominant over E-cadherin in relation to the aspects of mesenchymal transition assayed in this study.

Immediate and delayed effects of E-cadherin inhibition on gene regulation and cell motility in human epidermoid carcinoma cells.

The invasion suppressor protein, E-cadherin, plays a central role in epithelial cell-cell adhesion. Loss of E-cadherin expression or function in various tumors of epithelial origin is associated with a more invasive phenotype. In this study, by expressing a dominant-negative mutant of E-cadherin (Ec1WVM) in A431 cells, we demonstrated that specific inhibition of E-cadherin-dependent cell-cell adhesion led to the genetic reprogramming of tumor cells. In particular, prolonged inhibition of cell-cell adhesion activated expression of vimentin and repressed cytokeratins, suggesting that the effects of Ec1WVM can be classified as epithelial-mesenchymal transition. Both short-term and prolonged expression of Ec1WVM resulted in morphological transformation and increased cell migration though to different extents. Short-term expression of Ec1WVM up-regulated two AP-1 family members, c-jun and fra-1, but was insufficient to induce complete mesenchymal transition. AP-1 activity induced by the short-term expression of Ec1WVM was required for transcriptional up-regulation of AP-1 family members and down-regulation of two other Ec1WVM-responsive genes, S100A4 and igfbp-3. Using a dominant-negative mutant of c-Jun (TAM67) and RNA interference-mediated silencing of c-Jun and Fra-1, we demonstrated that AP-1 was required for cell motility stimulated by the expression of Ec1WVM. In contrast, Ec1WVM-mediated changes in cell morphology were AP-1-independent. Our data suggest that mesenchymal transition induced by prolonged functional inhibition of E-cadherin is a slow and gradual process. At the initial step of this process, Ec1WVM triggers a positive autoregulatory mechanism that increases AP-1 activity. Activated AP-1 in turn contributes to Ec1WVM-mediated effects on gene expression and tumor cell motility. These data provide novel insight into the tumor suppressor function of E-cadherin.