Biology subjects

Squires, I.

Publications and source records attributed to Squires, I..

6 recordsLinked to original sources

bioRxiv2026

Epithelial-Mesenchymal Transition Induces FAM134A-mediated ER-phagy to Regulate Procollagen Secretion

EMT is accompanied by extensive remodeling of the secretory pathway, yet how cancer cells maintain endoplasmic reticulum (ER) homeostasis during this transition remains unclear. Here we show that EMT activates ER-phagy as an adaptive mechanism to constrain secretory output. A genome-wide CRISPR activation screen unexpectedly identified EMT- and MET-associated transcription factors as opposing regulators of ER-phagy, and TGF{beta}-induced EMT robustly stimulated this pathway. Proteomic analysis revealed that ER-phagy regulates EMT-induced secretory protein degradation, including procollagens. Among FAM134 ER-phagy receptors, FAM134A uniquely mediated procollagen degradation through a non-canonical, LC3-independent mechanism. Loss of FAM134A diverted procollagen from degradation to secretion, resulting in a pro-invasive secretome, as conditioned media from FAM134A-deficient cells enhanced matrix invasion. These findings identify ER-phagy as a regulatory node that buffers EMT-associated secretory remodeling and reveal FAM134A as a context-specific suppressor of pro-invasive secretion.

cell biology↗

bioRxiv2026

NEK1 autophosphorylation is disrupted by amyotrophic lateral sclerosis-associated missense variants: activity biomarkers and structural insights

Rare variants in NEK1, encoding a serine/threonine kinase, are amongst the most consistently implicated genetic contributors to amyotrophic lateral sclerosis (ALS), reported in approximately 2-3% of cases. Yet, whilst recent studies have characterised the cell biological consequences of NEK1 loss-of-function, the biochemical effects of ALS-associated missense variants on kinase activity have not been directly investigated. This distinction is mechanistically important, because missense alleles encode mutant proteins rather than simply reducing protein dosage. Here, we provide the most comprehensive cell-based phosphoproteomic map of NEK1 phosphorylation to date, identifying ten recurrent phosphorylation sites across independent expression and acquisition conditions. We experimentally assign pSer14, pThr156 and pSer418 as NEK1 autophosphorylation sites using kinase-dead controls, targeted extracted ion chromatogram analysis, phosphosite mutagenesis and phosphospecific antibodies. Leveraging activation-loop pThr156 as a readout of NEK1 activity, we assessed nine ALS-associated missense variants spanning the major functional regions of the protein. Amongst catalytic-domain variants, R261C produced the most robust reduction in pThr156 autophosphorylation, R232C produced a smaller reduction, and R232H increased pThr156; the basic-region variant, A313T, also showed a smaller reduction. Structural modelling provides a mechanistic framework for understanding these variant-specific effects. Our study establishes the first activity-based framework for functional classification of NEK1 missense variants, and provides direct evidence that ALS-associated missense variants can alter NEK1 autophosphorylation through a mechanism distinct from simple haploinsufficiency. The phosphospecific antibodies, isogenic cell lines and curated phosphoproteomic datasets generated here provide a community resource for future studies of NEK1 regulation, variant interpretation and therapeutic target validation.

neuroscience↗

bioRxiv2025

Phosphorylation enables progressive microtubule-associated protein proteolysis and functionalisation during neural development

Neurological diseases are frequently characterised by dysregulation of the microtubule cytoskeleton, which is critical for neuronal integrity and functioning. However, the precise mechanisms by which microtubule dynamics are regulated during the development of the nervous system remain poorly understood. Here, we use global phosphoproteomic screening to identify cytoskeletal substrates of Ser-Arg Protein Kinase (SRPK), which is implicated in several neurodevelopmental and neurodegenerative conditions. We show that SRPK directly phosphorylates Microtubule Associated Protein (MAP)1S at multiple sites in a C-terminal region involved in proteolytic maturation and microtubule binding. SRPK-dependent MAP1S phosphorylation modulates the affinity of the MAP1S microtubule binding domain for microtubules and MAP1S proteolytic processing by the Calpain (CAPN)10 protease. Finally, we show that MAP1S proteolytic processing occurs progressively during neurodevelopment via a specific CAPN10 expression switch, corresponding with MAP1S acquisition of microtubule binding activity. Our results demonstrate a role for SRPK in coordinating processing and functionalisation of a key microtubule regulatory protein during neurodevelopment and provide insight into mechanisms by which the microtubule cytoskeleton may be dysregulated in neurological diseases.

biochemistry↗

bioRxiv2023

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in the C. elegans zygote that the feedback relies on aPKC phosphorylation of Cdc42 at serine 71. The turnover of CDC-42 phosphorylation ensures optimal aPKC asymmetry and activity throughout polarisation by tuning Par6/aPKC association with Par3 and Cdc42. Moreover, turnover of Cdc42 phosphorylation regulates actomyosin cortex dynamics that are known to drive aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust control of polarisation in many cell types. Key findings/graphical abstract- Phosphorylation of CDC-42 by aPKC accelerates aPKC dissociation from CDC-42, limiting aPKC activity - CDC-42/aPKC dissociation promotes aPKC association with PAR-3 and, thereby, aPKC asymmetry due to actomyosin flow - Cycling of CDC-42 phosphorylation fuels the exchange of aPKC between anteriorly transported PAR-3 and aPKC-active CDC-42 complexes - Turnover of CDC-42 phosphorylation alternates its association with effectors, aPKC and MRCK-1, ensuring proper actomyosin dynamics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/563985v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@e0ec65org.highwire.dtl.DTLVardef@c00b8dorg.highwire.dtl.DTLVardef@36565dorg.highwire.dtl.DTLVardef@160149a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination

As in mammals, only one cell in a Drosophila multicellular female germline cyst is specified as an oocyte. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here we demonstrate that Shots actin-binding domain (ABD) is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 (CH1) of Shots ABD recognises fusomal F-actin and requires CH2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of Utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

cell biology↗

bioRxiv2021

Symmetry breaking in the female germline cyst

In mammals and flies, only a limited number of cells in a multicellular female germline cyst become oocytes, but how the oocyte is selected is unknown. Here we show that the microtubule minus end-stabilizing protein, Patronin/CAMSAP marks the future Drosophila oocyte and is required for oocyte specification. The spectraplakin, Shot, recruits Patronin to the fusome, a branched structure extending into all cyst cells. Patronin stabilizes more microtubules in the cell with most fusome and this weak asymmetry is amplified by Dynein-dependent transport of Patronin-stabilized microtubules. This forms a polarized microtubule network, along which Dynein transports oocyte determinants into the presumptive oocyte. Thus, Patronin amplifies a weak fusome anisotropy to break cyst symmetry. These findings reveal a molecular mechanism of oocyte selection in the germline cyst.

developmental biology↗