Biology subjects

Karatas, Z.

Publications and source records attributed to Karatas, Z..

3 recordsLinked to original sources

bioRxiv2025

Using DNA origami to study nanoscale organization of plasma membranes

Plasma membrane (PM) lipids and proteins are organized into nanoscale regions called nanodomains, which regulate essential cellular processes by controlling local membrane organization. Despite advances in super-resolution microscopy and single particle tracking, the small size and temporal instability of nanodomains make them difficult to study in living cells. To overcome these challenges, we built fluorescent DNA origami probes that insert into the PM via lipid anchors displayed on the cell. The number and spatial distribution of anchors between the origami and the cell surface were precisely defined by the origami, enabling nanometer-scale sampling of the cell surface. Inserting these DNA origami particles into the membrane with lipid anchors allowed them to passively diffuse across the membrane, and we tracked their movement using single particle tracking to survey the PM landscape. By varying the number and spatial arrangement of lipid anchors connecting the DNA origami to the cell surface, we showed that immobilization of DNA origami particles requires simultaneous interactions with multiple nanodomains. Disruption of the actin cytoskeleton reduced immobilization, confirming its role in supporting nanodomain stability. Moreover, transient mechanical stretching of cells led to reversible increases in DNA origami mobility, indicating that mechanical force can reversibly regulate PM nanodomain organization. Altogether, we present a novel membrane-integrated DNA origami approach that provides mechanistic insights into PM nanodomain architecture and dynamics in living cells.

cell biology↗

bioRxiv2020

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

AO_SCPLOWBSTRACTC_SCPLOWFocal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown. Using single protein tracking, super-resolution microscopy and functional assays, we linked the molecular behavior and tridimensional nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displayed free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation during cell adhesion and spreading. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation.

cell biology↗

bioRxiv2017

Fast Activation Cycles Of Rac1 At The Lamellipodium Tip Trigger Membrane Protrusion

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE complex, effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Yet, correlation of Rho GTPases activation with cycles of membrane protrusions, suggested that Rac1 activation is not synchronized with membrane protrusion and occurs behind the lamellipodium. However, RhoA activation is maximal at the cell edge and synchronized with edge progression. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function of Rho GTPases mutants, we demonstrate that Rac1 immobilizations at the lamellipodium tip are correlated with Rac1 activation, on the contrary to RhoA. We show that Rac1 effector WAVE and Rac1 regulator IRSp53 accumulate at the lamellipodium tip by membrane free-diffusion and trapping. Nevertheless, wild-type Rac1, which directly interacts with WAVE and IRSp53, only displays slower diffusion at the lamellipodium tip, suggesting fast local activation/inactivation cycles. Local optogenetic activation of Rac1, triggered by Tiam1 membrane recruitment, proves that Rac1 activation must occur at the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. Furthermore, coupling tracking with optogenetic activation of Rac1 demonstrates that Rac1-WT diffusive properties are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model where Rac1 is rapidly switching between activation and inhibition at the lamellipodium tip, ensuring a local and fast control of Rac1 actions on its targets.\n\nSignificanceRac1 and RhoA GTPases are molecular switches controlling the actin cytoskeletal during cell migration. WAVE, Rac1 effector during cell protrusion, is concentrated at the lamellipodium tip. But, recent biosensor imaging studies suggested that Rac1 activation occurs behind the lamellipodium, while RhoA activation is maximal at the cell edge. Using single-molecule imaging and optogentics Rac1 activation we solved this apparent contradiction. We revealed a strong correlation between Rac1 activation and transient immobilizations at the lamellipodium tip, unlike RhoA. Furthermore, we demonstrated that Rac1 must be activated at the lamellipodium tip and not away from it to stimulate protrusion. Thus, fast cycling between activation and inhibition at the proximity of Rac1 targets ensures a local and fast control over Rac1 actions.\n\nAbbreviations

cell biology↗