Biology subjects

Sittewelle, M.

Publications and source records attributed to Sittewelle, M..

6 recordsLinked to original sources

bioRxiv2026

MicroRNA miR-219 is required for neural border and neural crest development in Xenopus neurulas

Neural crest (NC) multipotent stem cells give rise to many tissues including most of the peripheral nervous system, pigment cells and the craniofacial mesenchyme and skeleton. During gastrulation and early neurulation, cranial NC cells are specified in the ectoderm territory located between the anterior neural plate ectoderm and the future pre-placodal and lateral non-neural ectoderm. At the end of neurulation, NC cells undergo an epithelial-to-mesenchymal transition and migrate to various locations in the developing embryo where they differentiate. While the fine-tuning of NC specification is increasingly being elucidated, many questions remain, including how microRNAs may govern expression of gene programs during these processes. MicroRNAs are short non-coding 20-22 nucleotides-long RNAs which regulate gene expression through post-transcriptional repression. We have identified miR-219 as a candidate regulator of Xenopus NC development. Here, miR-219-dependent molecular pathways were investigated by morpholino knock-down and reveal NC phenotypes. The development of the NC and adjacent ectoderm was evaluated using whole mount in situ hybridization of key markers (pax3, zic1, xhe2, sox10, snai2, sox2), alcian blue cartilage staining, phenotype analysis, RNA sequencing of microdissected dorsal ectoderm and microRNA rescue experiments. While neural induction is mainly unaffected, miR-219 depletion alters gene expression programs associated with neural border development, resulting in loss of NC specification. HighlightsO_LImiR-219 depletion expands the neural border territory and disrupts neural crest specification. C_LIO_LImiR-219 depletion phenotypes are rescued with miRNA mimics. C_LIO_LImiR-219 morphant neural border expansion is rescued by pax3 depletion. C_LIO_LIRNA-seq reveals specific gene program modulation in miR-219 morphant neural crest. C_LIO_LImiR-219 is predicted to directly downregulate the neural gene Hes5.3. C_LI

developmental biology↗

bioRxiv2025

MicroRNA miR-196a controls neural crest patterning by repressing immature neural ectoderm programs in Xenopus embryos.

HighlightsmiR-196a promotes Neural Crest development: O_LIRNA sequencing of the developing neural border and neural crest reveals miR-196a-dependent gene programs. C_LIO_LImiR-196a represses early neural plate programs and reduces pluripotency gene expression. C_LIO_LImiR-196a may directly control neural crest gene sox10 in vivo. C_LI Neural crest (NC) cells form a multipotent stem cell population specified during neurulation, which undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively in the developing embryo, to generate numerous tissues and cell types including the craniofacial skeleton, the peripheral nervous system and pigment cells. The genetic and molecular details of NC specification are governed by a complex, yet still partially understood gene regulatory network (NC-GRN). In particular, the precise function of microRNAs (miRNA) in this network remains poorly characterized. miRNAs are short non-coding 20-22 nucleotides long RNAs, which control gene expression through post-transcriptional repression. Since miRNA-196a is expressed in the developing neural and NC cells of Xenopus laevis embryos, we here investigated miR-196a function in the NC-GRN, by knocking-down its expression using antisense morpholinos. Depletion of miR-196a revealed major NC and craniofacial phenotypes. These defects were preceded by the perturbed expression of key neural, neural border and NC markers such as sox2/3, zic1/3, pax3, sox10 and snail2. Using RNA sequencing of individual neural border and NC explants, we have identified a signature of genes up- and down-regulated by miR-196a and validate these with rescue experiments using a miRNA mimic. Our study identifies miR-196a as a key actor of early patterning in the dorsal ectoderm, balancing the extent of immature neural plate progenitors with NC and placode specification, while also promoting neuron differentiation within the neural plate.

developmental biology↗

bioRxiv2023

Passive diffusion accounts for the majority of intracellular vesicle transport

During membrane trafficking, a vesicle formed at the donor compartment must travel to the acceptor membrane before fusing. For large carriers, it is established that this transport is motor-driven; however, the mode by which small vesicles, which outnumber larger carriers, are transported is poorly characterized. Here we show that intracellular nanovesicles (INVs), a substantial class of small vesicles, are highly mobile within cells and that this mobility depends almost entirely on passive diffusion (0.1-0.3 {micro}m2 s-1). Using single particle tracking, we describe how other small trafficking vesicles have a similar diffusive mode of transport, that contrasts with the motor-dependent movement of larger endolysosomal carriers. We also demonstrate that a subset of INVs are involved in constitutive secretion and that delivery of cargo to the plasma membrane during exocytosis is decreased when diffusion of INVs is specifically restricted. Our results suggest that passive diffusion is sufficient to explain the majority of small vesicle transport.

cell biology↗

bioRxiv2022

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation

The formation of a clathrin-coated vesicle is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment "hook" to an "anchor" protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin alone is sufficient for intracellular clathrin-coated vesicle formation.

cell biology↗

bioRxiv2020

The glycolysis regulator PFKFB4 interacts with ICMTand activates RAS/AKT signaling-dependent cell migration in melanoma.

Cell migration is a complex process, tightly regulated during embryonic development and abnormally activated during cancer metastasis. RAS-dependent signaling is a major nexus controlling essential cell parameters including proliferation, survival and migration, utilizing downstream effectors such as the PI3K/AKT signaling pathway. In melanoma, oncogenic mutations frequently enhance RAS, PI3K/AKT or MAP kinase signaling, and trigger other cancer hallmarks among which the activation of metabolism regulators. PFKFB4 is one of these critical regulators of glycolysis and of the Warburg effect. Here however, we explore a novel function of PFKFB4 in melanoma cell migration. We find that PFKFB4 interacts with ICMT, a post-translational modifier of RAS. PFKFB4 promotes ICMT/RAS interaction, controls RAS localization at the plasma membrane, activates AKT signaling and enhances cell migration. We thus provide evidence of a novel and glycolysis-independent function of PFKFB4 in human cancer cells. This unconventional activity links the metabolic regulator PFKFB4 to RAS-AKT signaling and impacts melanoma cell migration. Highlights- PFKFB4, a known regulator of glycolysis, also displays an unconventional role in melanoma cell migration. - PFKFB4 interacts with ICMT and promotes RAS localization at the plasma membrane. - PFKFB4 and ICMT cooperation modulates AKT signaling and controls melanoma cell migration.

cell biology↗

bioRxiv2017

PFKFB4 control of Akt signaling is essential for premigratory and migratory neural crest formation.

Summary statementPFKFB4 controls neural crest final specification and migration by regulation of AKT signaling or glycolysis.\n\nAbstractNeural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting into functional premigratory NC, able to delaminate and migrate. We found that the NC-GRN triggers up-regulation of pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions in vivo. We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail activating n-cadherin and late NC specifiers, exhibit severe migration defects, resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, while both AKT signaling and glycolysis regulate migration. These findings highlight novel and critical roles of PFKFB4 activity in later stages of NC development, wired into the NC-GRN.

developmental biology↗