Nesprin-1α-Dependent Microtubule Nucleation from the Nuclear Envelope via Akap450 Is Necessary for Nuclear Positioning in Muscle Cells

Abstract

SummaryThe nucleus is the main microtubule-organizing center (MTOC) in muscle cells due to the accumulation of centrosomal proteins and microtubule (MT) nucleation activity at the nuclear envelope (NE) [1, 2, 3, 4]. The relocalization of centrosomal proteins, including Pericentrin, Pcm1, and γ-tubulin, depends on Nesprin-1, an outer nuclear membrane (ONM) protein that connects the nucleus to the cytoskeleton via its N-terminal region [5, 6, 7]. Nesprins are also involved in the recruitment of kinesin to the NE and play a role in nuclear positioning in skeletal muscle cells [8, 9, 10, 11, 12]. However, a function for MT nucleation from the NE in nuclear positioning has not been established. Using the proximity-dependent biotin identification (BioID) method [13, 14], we found several centrosomal proteins, including Akap450, Pcm1, and Pericentrin, whose association with Nesprin-1α is increased in differentiated myotubes. We show that Nesprin-1α recruits Akap450 to the NE independently of kinesin and that Akap450, but not other centrosomal proteins, is required for MT nucleation from the NE. Furthermore, we demonstrate that this mechanism is disrupted in congenital muscular dystrophy patient myotubes carrying a nonsense mutation within the SYNE1 gene (23560 G>T) encoding Nesprin-1 [15, 16]. Finally, using computer simulation and cell culture systems, we provide evidence for a role of MT nucleation from the NE on nuclear spreading in myotubes. Our data thus reveal a novel function for Nesprin-1α/Nesprin-1 in nuclear positioning through recruitment of Akap450-mediated MT nucleation activity to the NE.

Atrial structural remodeling gene variants in patients with atrial fibrillation

“…AKAP9, a scaffolding protein involved in Golgi apparatus integrity and Golgi-related microtubules nucleation [35], is known to be the long QT syndrome-causative gene [49]. Our results confirmed an altered microtubule network in absence of AKAP9 as inhibition of AKAP9 results in increased aggregation phenotype in myotubes [36]. Consequences of AKAP9 knockdown on remaining pool of microtubule-associatepartners remain to be determines.…”

“…using C2C12 myoblast and quantify myonuclei aggregation in AKAP9-depleted myotubes using 3 different siRNA (Figure 4). AKAP9-depleted myotubes significantly increase myonuclei aggregation phenotype (up to 30%) within myotubes (Figure 4(c)) without affecting myoblast fusion or myotubes differentiation (Figures 4(a) and 4(b)), confirming a microtubule integrity regulation by an AKAP9-dependant mechanism in a muscle cells context [36].…”

“…It has been recently shown that AKAP9 can contribute to recruit microtubule-organizing center factors at the membrane of myonuclei [36]. We validated AKAP9-dependent myonuclei positioning in a muscle cells context Specific standard terminologies-"pathogenic", "likely pathogenic", "uncertain significance", "likely benign" and "benign" were used to describe variants identified [28] The presence of an (X) indicates involvement of the gene in each specific cardiac disease classification.…”

The dual role of the centrosome in organizing the microtubule network in interphase

“…Inactivation of the centrosome is usually accompanied by shedding PCM components such as γ‐tubulin, PCNT, CDK5Rap2, or AKAP450. Interestingly, these proteins are associated with Golgi membranes in both differentiated cells and in centrinone‐induced centrosome‐less cells (see herein). Again, in both cases, MT nucleation was driven by AKAP450, whereas CDK5Rap2 and, especially, PCNT, had no significant influence .…”

“…Interestingly, these proteins are associated with Golgi membranes in both differentiated cells and in centrinone‐induced centrosome‐less cells (see herein). Again, in both cases, MT nucleation was driven by AKAP450, whereas CDK5Rap2 and, especially, PCNT, had no significant influence . These results suggest that centrinone‐treated cells mimic the behavior of naturally occurring centrosome‐inactivated differentiated cells.…”