Biology subjects

Dema, A.

Publications and source records attributed to Dema, A..

5 recordsLinked to original sources

bioRxiv2024

α-Synuclein acts as a cholesteryl-ester sensor on lipid droplets regulating organelle size and abundance.

While aggregated alpha-Synuclein (Syn) is commonly associated with Parkinsons disease, its physiological function as a membrane-binding protein is poorly understood. Here, we show that endogenous Syn binds lipid droplets (LDs) in multiple human cell lines and in stem cell-derived dopaminergic neurons. LD-binding encompasses Syn residues 1-100, which masks their detection by immunofluorescence microscopy, probably explaining the scarcity of similar observations in earlier studies. Syn-LD interactions are highly temperature-sensitive and selective for cholesteryl-ester-rich LDs. They promote the formation of Syn multimers that dissociate from LDs at non-permissive temperatures. Syn remains LD-bound throughout starvation-induced lipolysis, whereas siRNA-knockdown diminishes LD abundance and compromises cell viability upon nutrient depletion, without affecting LD biosynthesis. Reciprocally, excess Syn stimulates LD accumulation in dependence of lipid availability, restricts organelle size and ensures intracellular LD organization, which strictly depends on functional membrane-binding. Supporting a general role of Syn in cellular lipid and cholesterol metabolism, our results point to additional loss-of-function similarities between Parkinsons, Alzheimers and Gauchers disease.

cell biology↗

bioRxiv2024

Doublecortin reinforces microtubules to promote growth cone advance in soft environments

Doublecortin (DCX) is a microtubule-associated protein critical for brain development. Although most highly expressed in the developing central nervous system, the molecular function of DCX in neuron morphogenesis remains unknown and controversial. We demonstrate that DCX function is intimately linked to its microtubule-binding activity. By using human induced pluripotent stem cell (hiPSC)- derived cortical i3Neurons genome engineered to express mEmerald-tagged DCX from the endogenous locus, we find that DCX-MT interactions become highly polarized very early during neuron morphogenesis. DCX becomes enriched only on straight microtubules in advancing growth cones with approximately 120 DCX molecules bound per micrometer of growth cone microtubule. At a similar saturation, microtubule-bound DCX molecules begin to impede lysosome transport, and thus can potentially control growth cone organelle entry. In addition, by comparing control, DCX-mEmerald and knockout DCX -/Y i3Neurons, we find that DCX stabilizes microtubules in the growth cone peripheral domain by reducing the microtubule catastrophe frequency and the depolymerization rate. DCX -/Y i3Neuron morphogenesis was inhibited in soft microenvironments that mimic the viscoelasticity of brain tissue and DCX -/Y neurites failed to grow toward brain-derived neurotrophic factor (BDNF) gradients. Together with high resolution traction force microscopy data, we propose a model in which DCX-decorated, rigid growth cone microtubules provide intracellular mechanical resistance to actomyosin generated contractile forces in soft physiological environments in which weak and transient adhesion-mediated forces in the growth cone periphery may be insufficient for productive growth cone advance. These data provide a new mechanistic understanding of how DCX mutations cause lissencephaly-spectrum brain malformations by impacting growth cone dynamics during neuron morphogenesis in physiological environments.

cell biology↗

bioRxiv2022

Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step enabling this approach in human induced pluripotent stem cells and derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance.

cell biology↗

bioRxiv2021

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, {pi}-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute {pi}-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, {pi}-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of {pi}-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

cell biology↗

bioRxiv2020

α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels.

The Parkinsons disease protein -synuclein (Syn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of Syn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate plasma membrane localization of endogenous pools of Syn in A2780, HeLa, SH-SY5Y and SK-MEL-2 cells. We demonstrate that Syn binds reconstituted PIP2-membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute Syn in cells. We further delineate that Syn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to Syns cellular function(s) at the plasma membrane.

cell biology↗