Caren Norden

The NoCut Pathway Links Completion of Cytokinesis to Spindle Midzone Function to Prevent Chromosome Breakage

During anaphase, spindle elongation pulls sister chromatids apart until each pair is fully separated. In turn, cytokinesis cleaves the cell between the separated chromosomes. What ensures that cytokinesis proceeds only after that all chromosome arms are pulled out of the cleavage plane was unknown. Here, we show that a signaling pathway, which we call NoCut, delays the completion of cytokinesis in cells with spindle-midzone defects. NoCut depends on the Aurora kinase Ipl1 and the anillin-related proteins Boi1 and Boi2, which localize to the site of cleavage in an Ipl1-dependent manner and act as abscission inhibitors. Inactivation of NoCut leads to premature abscission and chromosome breakage by the cytokinetic machinery and is lethal in cells with spindle-elongation defects. We propose that NoCut monitors clearance of chromatin from the midzone to ensure that cytokinesis completes only after all chromosomes have migrated to the poles.

Actomyosin Is the Main Driver of Interkinetic Nuclear Migration in the Retina

Summary Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.

Uncovering multiple axonal targeting pathways in hippocampal neurons

Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins.

A mechanism for chromosome segregation sensing by the NoCut checkpoint

In Saccharomyces cerevisiae and HeLa cells, the NoCut checkpoint, which involves the chromosome passenger kinase Aurora B, delays the completion of cytokinesis in response to anaphase defects. However, how NoCut monitors anaphase progression has not been clear. Here, we show that retention of chromatin in the plane of cleavage is sufficient to trigger NoCut, provided that Aurora/Ipl1 localizes properly to the spindle midzone, and that the ADA histone acetyltransferase complex is intact. Furthermore, forcing Aurora onto chromatin was sufficient to activate NoCut independently of anaphase defects. These findings provide the first evidence that NoCut is triggered by the interaction of acetylated chromatin with the passenger complex at the spindle midzone.

Ankyrin-dependent and -independent mechanisms orchestrate axonal compartmentalization of L1 family members neurofascin and L1/neuron-glia cell adhesion molecule.

Axonal initial segments (IS) and nodes of Ranvier are functionally important membrane subdomains in which the clustering of electrogenic channels enables action potential initiation and propagation. In addition, the initial segment contributes to neuronal polarity by serving as a diffusion barrier. To study the mechanisms of axonal compartmentalization, we focused on two L1 family of cell adhesion molecules (L1-CAMs) [L1/neuron–glia cell adhesion molecule (L1/NgCAM) and neurofascin (NF)] and two neuronal ankyrins (ankB and ankG). NF and ankG accumulate specifically at the initial segment, whereas L1/NgCAM and ankB are expressed along the entire lengths of axons. We find that L1/NgCAM and NF show distinct modes of steady-state accumulation during axon outgrowth in cultured hippocampal neurons. Despite their different steady-state localizations, both L1/NgCAM and NF show slow diffusion and low detergent extractability specifically in the initial segment but fast diffusion and high detergent extractability in the distal axon. We propose that L1-CAMs do not strongly bind ankB in the distal axon because of spatial regulation of ankyrin affinity by phosphorylation. NF, conversely, is initially enriched in an ankyrin-independent manner in the axon generally and accumulates progressively in the initial segment attributable to preferential binding to ankG. Our results suggest that NF and L1/NgCAM accumulate in the axon by an ankyrin-independent pathway, but retention at the IS requires ankyrin binding.

Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia

Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics – the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.

Mechanisms controlling arrangements and movements of nuclei in pseudostratified epithelia

During development, cells undergo complex rearrangements that contribute to the final tissue architecture. A characteristic arrangement found in rapidly expanding, highly proliferative tissues is pseudostratified epithelium, which features notably elongated cells with varied nuclear positions along the cell axis. Although anomalies in its structure are implicated in diseases like microcephaly, how pseudostratification is formed and maintained remains elusive. In this review, we focus on a typical feature of pseudostratified epithelia called interkinetic nuclear migration (INM), which describes dynamic movements of nuclei within the elongated cell bodies. We provide an overview of cytoskeletal components underlying INM in different systems, discuss current understanding of its kinetics and timing, and evaluate how conflicting results could be explained through developmental and evolutionary considerations.

Interkinetic Nuclear Migration Is Centrosome Independent and Ensures Apical Cell Division to Maintain Tissue Integrity

Pseudostratified epithelia are widespread during animal development and feature elongated cells whose nuclei adopt various positions along the apicobasal cell axis. Before mitosis, nuclei migrate toward the apical surface, and subsequent divisions occur apically. So far, the exact purpose of this nuclear migration remained elusive. One hypothesis was that apical migration ensures that nuclei and centrosomes meet for mitosis. We here demonstrate that in zebrafish neuroepithelia apical nuclear migration occurs independently of centrosome position or integrity. It is a highly reproducible phenomenon linked to the cell cycle via CDK1 activity. We propose that the robustness of bringing nuclei apically for mitosis ensures that cells are capable of reintegrating into the epithelium after division. Nonapical divisions lead to cell delamination and formation of cell clusters that subsequently interfere with neuronal layering. Therefore, positioning divisions apically in pseudostratified neuroepithelia could serve to safeguard epithelial integrity and enable proper proliferation and maturation.

The vertebrate retina: A model for neuronal polarization in vivo

The vertebrate retina develops rapidly from a proliferative neuroepithelium into a highly ordered laminated structure, with five distinct neuronal cell types. Like all neurons, these cells need to polarize in appropriate orientations order integrate their neuritic connections efficiently into functional networks. Its relative simplicity, amenability to in vivo imaging and experimental manipulation, as well as the opportunity to study varied cell types within a single tissue, make the retina a powerful model to uncover how neurons polarize in vivo. Here we review the progress that has been made thus far in understanding how the different retinal neurons transform from neuroepithelial cells into mature neurons, and how the orientation of polarization may be specified by a combination of pre‐established intrinsic cellular polarity set up within neuroepithelial cells, and extrinsic cues acting upon these differentiating neurons.

Slit1b-Robo3 Signaling and N-Cadherin Regulate Apical Process Retraction in Developing Retinal Ganglion Cells

When neurons exit the cell cycle after their terminal mitosis, they detach from the apical surface of the neuroepithelium. Despite the fact that this detachment is crucial for further neurogenesis and neuronal migration, the underlying mechanisms are still not understood. Here, taking advantage of the genetics and imaging possibilities of the zebrafish retina as a model system, we show by knockdown experiments that the guidance molecule Slit1b and its receptor Robo3 are required for apical retraction of retinal ganglion cells (RGCs). In contrast, N-cadherin seems to be responsible for maintenance of apical attachment, as expression of dominant-negative N-cadherin causes RGCs to lose apical attachments prematurely and rescues retraction in slit1b morphants. These results suggest that Slit-Robo signaling downregulates N-cadherin activity to allow apical retraction in newly generated RGCs.