Biology subjects

Fantner, G. E.

Publications and source records attributed to Fantner, G. E..

13 recordsLinked to original sources

bioRxiv2025

CDK4 Restricts Triple-Negative Breast Cancer Cell Migration via Phosphorylation-Driven Activation of Myo9b RhoGAP Function

Cyclin-Dependent Kinase 4 (CDK4) is a key regulator of cell cycle progression, driving the G0/G1-to-S phase transition through phosphorylation of Retinoblastoma 1 (RB1). Clinically, CDK4/6 inhibitors are under investigation in Triple Negative Breast Cancer (TNBC), a subtype characterized by invasiveness, aggressiveness and poor prognosis. While CDK4 is primarily targeted for its role in proliferation, emerging evidence suggests it may also regulate other cellular processes. In particular, the mechanisms by which CDK4 could influence cancer cell migration, remain largely unexplored, particularly in highly heterogenous cell line like MDA-MB-231. This study investigates whether CDK4 contributes to the regulation of TNBC cells migration and identifies the pathways involved in MDA-MB-231 cells, independently of its role in proliferation. We demonstrate that loss or inhibition of CDK4, using respectively CRISPR/Cas9 mediated CDK4 knockout and pharmacological CDK4/6 inhibitor, leads to enhanced migration capacities and reorganization of actin subcellular networks. Mechanistically, the absence of CDK4 results in decreased phosphorylation of Myo9b at serine 1935 (S1935), which enhances RhoA signaling, a key driver of cytoskeletal dynamics, leading to polarity defects and increased cell migration. These findings reveal a non-canonical function of CDK4 in limiting TNBC cell migration through the CDK4/CyclinD-Myo9b-RhoA signaling axis. This work highlights the broader cellular roles of CDK4 beyond its established function in proliferation and suggest that inhibition of Myo9b-RhoA pathway could reduce metastatic behaviour in TNBC treated with CDK4/6i, thereby informing future co-therapeutic strategies against aggressive cancer subtypes.

cell biology↗

bioRxiv2025

Separate-scan atomic force microscope for fast infrared scattering-type scanning near-field optical microscope

Pseudo-heterodyne scattering-type scanning near-field optical microscopy (sSNOM) is applied in the mid-infrared region to detect the chemical composition of biomolecules on the nanoscale. However, the application of sSNOM in molecular biology has been limited to static images in air. Recently, bottom illumination sSNOM (BI-sSNOM) was developed for operation in water. Yet, the scan rate of sSNOM remains a bottleneck to record protein structural changes in aqueous solution on the seconds time scale. We designed an optical and mechanical system consisting of a separate scan high-speed atomic force microscope (HS-AFM) coupled to the BI-sSNOM optics. The designed AFM scanner has a mechanical bandwidth of ca 70 kHz along the Z-axis, and ca 6 kHz along the XY-axis, equivalent to the sample scanning HS-AFM. The AFM performance is demonstrated by imaging actin filaments. The optical design is validated by sSNOM experiments on purple membranes and microtubules.

biophysics↗

bioRxiv2025

Deep-learning-assisted SICM for enhanced real-time imaging of nanoscale biological dynamics

Scanning Ion Conductance Microscopy (SICM) provides high-resolution, nanoscale imaging of living cells, but it is generally limited by a slow scan rate, making it challenging to capture dynamic processes in real time. To tackle this challenge, we propose an integrated data acquisition and computational framework that improves the temporal resolution of SICM by selectively skipping certain scan lines. A partial convolutional neural network (Partial-CNN) model is developed and trained on SICM images and their corresponding masks to reconstruct the complete images from the under-sampled data, ensuring the retention of structural integrity. This approach significantly reduces the image acquisition time (i.e., by 30-60%) without compromising quality, as validated through multiple quantitative metrics. Compared to conventional deep learning methods, the Partial-CNN demonstrates higher accuracy in reconstructing fine details and maintaining consistent height maps across skipped regions. We show that this method provides an increased temporal resolution and retains image fidelity, making it suitable for real-time dynamic SICM imaging and improving the smart scanning microscopy applications in time-resolved biological imaging. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/663539v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@562d32org.highwire.dtl.DTLVardef@1b12dcaorg.highwire.dtl.DTLVardef@13535f3org.highwire.dtl.DTLVardef@6904f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

A hermetically closed sample chamber enables time-lapse nano-characterization of pathogenic microorganisms in-vitro

Pathogenic microorganisms, such as pathogenic mycobacteria, pose a global health burden. Studying these organisms is crucial for gaining detailed knowledge about the pathogens and the diseases they cause. To handle pathogenic organisms, specific biosafety measures appropriate to the virulence of the organism must be fulfilled, most importantly ensuring that all manipulations of pathogenic material are performed within a confined environment. Atomic force microscopy (AFM) is a powerful technique to study biological samples at nanometer-scale resolution, yielding also mechanical properties, all while maintaining physiological conditions. However, standard AFM sample holders do not meet stringent biosafety requirements since they do not constitute a confined system. AFM imaging relies on direct contact between the cantilever and the sample and is sensitive to mechanical interference, rendering conventional containment systems for handling infectious substances inapplicable. Here, we introduce a hermetically sealed AFM sample chamber that meets biosafety demands while satisfying the mechanical and optical constraints of correlated optical microscopy and AFM. We imaged various pathogenic mycobacteria to demonstrate the chambers versatility and effectiveness in containing biohazardous materials. This sample chamber enables high-resolution, time-lapse correlated imaging and biomechanical characterization of pathogenic microorganisms in-vitro. It broadens the scope of research with pathogenic microorganisms under safe and controlled conditions.

bioengineering↗

bioRxiv2023

Structural flexibility dominates over binding strength for supramolecular crystallinity

Supramolecular crystallinity is abundantly present in nature and results from directional, weak non-covalent interactions between components. Bottom-up nanotechnology aims to exploit such phenomena to control the self-assembly of ordered networks and complex objects from rationally designed monomers. Like all crystalline materials, 2D supramolecular crystals develop from an initial nucleation site, followed by growth, based on directional interactions. Traditionally, the binding strength and directionality of interactions is thought to dictate the nucleation and crystal growth, whereas structural flexibility favours defects. Usually, macromonomers present multiple binding units with relative intramolecular flexibility that affects their intermolecular interactions. Thus far, the effects of such flexibility on supramolecular assembly have not been explored. Here we introduce the concept of "interface flexibility" and demonstrate its critical importance in the nucleation and growth of supramolecular crystalline networks. We show that tuning the interface flexibility greatly expands the available design space for synthetic supramolecular crystalline materials.

bioengineering↗

bioRxiv2023

OpenSIM: open source microscope add-on for structured illumination microscopy

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

bioengineering↗

bioRxiv2022

Spatially Multiplexed Single-molecule Translocations through a Nanopore at Controlled Speeds

Nanopores are one of the most successful label-free single-molecule techniques with several sensing applications such as biological screening, diagnostics, DNA and protein sequencing1-4. In current nanopore technologies, stochastic processes influence both the selection of the translocating molecule, translocation rate and translocation velocity5,6. As a result, single-molecule translocations are difficult to control spatially and temporally. Here we present a novel method where we engineer precise spatial and temporal control into the single-molecule experiment. We use a glass nanopore mounted on a 3D nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and the glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio (SNR) by two orders of magnitude compared to free translocations. We applied our method to various DNA constructs, achieving down to single nucleotide gap resolution. The spatial multiplexing combined with the sub-nanometer resolution could be used in conjunction with micro-array technologies to enable screening of DNA, improving point of care devices, or enabling high-density, addressable DNA data storage.

biophysics↗

bioRxiv2022

Mechanical morphotype switching as an adaptive response in mycobacteria

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report a new virulence mechanism by which mycobacteria alter the mechanical stiffness of their cell surface to become refractory to killing during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Genetic manipulation, by deletion of uvrA, or pharmacological treatment, with bedaquiline, locked mycobacteria into a "soft" mechanical morphotype state, enhancing survival in macrophages. Our study proposes microbial mechanical adaptation as a new axis for surviving host-mediated stressors. One-Sentence SummaryBacteria alter their cell surface mechanical properties to increase survival during macrophage infection.

microbiology↗

bioRxiv2021

Time-resolved scanning ion conductance microscopy for three-dimensional tracking of nanoscale cell surface dynamics

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy1, EM2, AFM3). However, time-resolved imaging techniques tend to either have lower resolution, are limited to small areas, or cause damage to the cells thereby preventing long-term time-lapse studies. Scanning probe microscopy methods such as atomic force microscopy (AFM) combine high-resolution imaging with the ability to image living cells in physiological conditions. The mechanical contact between the tip and the sample, however, deforms the cell surface, disturbs the native state, and prohibits long-term time-lapse imaging. Here, we develop a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging. By utilizing recent advances in nanopositioning4, nanopore fabrication5, microelectronics6, and controls engineering7 we developed a microscopy method that can resolve spatiotemporally diverse three-dimenional processes on the cell membrane at sub-5nm axial resolution. We tracked dynamic changes in live cell morphology with nanometer details and temporal ranges of sub-second to days, imagining diverse processes ranging from endocytosis, micropinocytosis, and mitosis, to bacterial infection and cell differentiation in cancer cells. This technique enables a detailed look at membrane events and may offer insights into cell-cell interactions for infection, immunology, and cancer research.

biophysics↗

bioRxiv2020

Correlative 3D microscopy of single cells using super-resolution and scanning ion-conductance microscopy

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffractional resolution.

biophysics↗

bioRxiv2020

Surface-catalyzed SAS-6 self-assembly directs centriole formation through kinetic and structural mechanisms

Discovering the physical principles directing organelle assembly is a fundamental pursuit in biology. Centrioles are evolutionarily conserved organelles with a 9-fold rotational symmetry of chiral microtubules imparted onto the cilia they template1. Centriole assemble from likewise symmetrical ring polymers of SAS-6 proteins, orthogonal to a toroidal surface surrounding the resident centriole2-4. How surface properties ensure ring assembly with proper symmetry and orthogonal arrangement is not known. Here, we deployed photothermally-actuated off-resonance tapping high-speed atomic force microscopy (PORT-HS-AFM) to decipher physical principles of surface-guided SAS-6 self-assembly. Using machine learning to quantify the polymerization reaction and developing a coagulation-fragmentation model, we discovered that the surface shifts the reaction equilibrium by [~]104 compared to the solution situation, explaining orthogonal organelle emergence. Moreover, molecular dynamics and PORT-HS-AFM revealed that the surface converts helical SAS-6 polymers into 9-fold ring polymers with residual asymmetry, which may impart chiral features to centrioles and cilia. Overall, we discovered two fundamental physical principles directing robust centriole organelle assembly.

biophysics↗

bioRxiv2018

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b6 interacts with the lipid bilayer prior to recruiting C7 and C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.

biophysics↗

bioRxiv2018

Maturing Mycobacterial Peptidoglycan Required Non-canonical Crosslinks to Maintain Shape

In most well studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin binding proteins (PBPs). However, in mycobacteria, L,D-transpeptidase (LDT)-mediated crosslinks are highly abundant. To elucidate the role of these unusual crosslinks, we characterized mycobacterial cells lacking all LDTs. We find that LDT-mediated crosslinks are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT-inhibition. Thus, knowledge about the single-cell distribution of drug targets can be exploited to more effectively treat this pathogen.

microbiology↗