CYLD regulates spindle orientation by stabilizing astral microtubules and promoting dishevelled-NuMA-dynein/dynactin complex formation

Abstract

Oriented cell division is critical for cell fate specification, tissue organization, and tissue homeostasis, and relies on proper orientation of the mitotic spindle. The molecular mechanisms underlying the regulation of spindle orientation remain largely unknown. Herein, we identify a critical role for cylindromatosis (CYLD), a deubiquitinase and regulator of microtubule dynamics, in the control of spindle orientation. CYLD is highly expressed in mitosis and promotes spindle orientation by stabilizing astral microtubules and deubiquitinating the cortical polarity protein dishevelled. The deubiquitination of dishevelled enhances its interaction with nuclear mitotic apparatus, stimulating the cortical localization of nuclear mitotic apparatus and the dynein/dynactin motor complex, a requirement for generating pulling forces on astral microtubules. These findings uncover CYLD as an important player in the orientation of the mitotic spindle and cell division and have important implications in health and disease.O rientation of the cell division axis offers a critical mechanism for the control of cell type choices and the specification of tissue/organ architecture; this is achieved through accurate orientation of the mitotic spindle relative to the cell cortex (1). Spindle orientation is exquisitely regulated during development as well as in adult life, and defects in this process may have severe consequences, such as developmental disorders and tumor formation (1, 2). A dividing cell can orient its spindle along the planar axis or the apicobasal axis of the tissue, depending on the tissue environment and cell geometry. In most epithelia, such as the intestine crypt epithelium, planar spindle orientation is common to produce two daughter cells side by side. By contrast, apicobasal spindle orientation is frequently associated with asymmetric cell divisions, which result in two daughter cells of distinct identities (2).Astral microtubules play a key role in spindle orientation by linking the spindle to the cell cortex (3). The localization of cell polarity proteins such as dishevelled (Dvl) at the cell cortex is also important for spindle orientation by transmission of extrinsic signals or providing the intrinsic cues. Cortical polarity proteins can recruit the nuclear mitotic apparatus (NuMA) protein and then the microtubule minus end-directed dynein/ dynactin motor complex, which can generate pulling forces on astral microtubules to rotate the spindle (3). Therefore, the dynamic interaction of astral microtubules with the cell cortex via diverse protein complexes constitutes an essential part of the mechanism for spindle orientation. However, it remains elusive how the protein complexes controlling spindle orientation are assembled and activated to make a connection between astral microtubules and the cell cortex.As a posttranslational modification, protein ubiquitination is critical for diverse cellular and biological events, and it is a reversal process mediated by E3 ubiquitin ligases and deubiquitinases, respecti...

CYLD mediates ciliogenesis in multiple organs by deubiquitinating Cep70 and inactivating HDAC6

“…TSA, tubacin, sodium butyrate, CAY10603, and tubastatin A were from Santa Cruz Biotechnology, and DAPI was from Sigma-Aldrich. The sequences for control and CYLD siRNAs were described previously [12]. Mammalian expression plasmids for GFP-CYLD, GFP-Cep70, GST-Cep70, HA-HDAC6, and His-Myc-ubiquitin were described previously [12,20,35], and various mutants were generated by PCR and site-directed mutagenesis.…”

“…The sequences for control and CYLD siRNAs were described previously [12]. Mammalian expression plasmids for GFP-CYLD, GFP-Cep70, GST-Cep70, HA-HDAC6, and His-Myc-ubiquitin were described previously [12,20,35], and various mutants were generated by PCR and site-directed mutagenesis. MBP-Cep70 was purified from bacteria expressing the pMALp2T-Cep70 plasmid as described previously [20], with amylose resin (New England Biolabs).…”

“…All MEFs were cultured in DMEM medium supplemented with 10% fetal bovine serum. To induce ciliary formation, RPE-1 cells were cultured in serum-free medium for 48 h. Cells were synchronized at G1, S, G2, or M phase as described previously [12]. Plasmids were transfected to cells using TurboFect (Thermo Scientific), and siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen).…”

“…CYLD also contains cytoskeleton-associated protein glycine-rich (CAP-Gly) domains that mediate its interaction with microtubules and histone deacetylase 6 (HDAC6) [10][11][12]. Several cilium-related symptoms, such as male infertility, osteoporosis, and impaired lung maturation, have been reported in CYLD knockout mice or in mice carrying truncated CYLD [13][14][15].…”

CYLD – a deubiquitylase that acts to fine-tune microtubule properties and functions

“…3). CYLD has been shown to be upregulated during mitosis (Stegmeier et al, 2007;Yang et al, 2014a). In HeLa cells, CYLD depletion delays mitotic entry in a deubiquitylase-activity-dependent manner, likely due to decreased activation of polo-like kinase 1 (PLK1) (Stegmeier et al, 2007) (Fig.…”