11 research outputs found
BMPâ2 signaling and mechanotransduction synergize to drive osteogenic differentiation via YAP/TAZ
Growth factors and mechanical cues synergistically affect cellular functions, triggering a variety of signaling pathways. The molecular levels of such cooperative interactions are not fully understood. Due to its role in osteogenesis, the growth factor bone morphogenetic protein 2 (BMPâ2) is of tremendous interest for bone regenerative medicine, osteoporosis therapeutics, and beyond. Here, contribution of BMPâ2 signaling and extracellular mechanical cues to the osteogenic commitment of C2C12 cells is investigated. It is revealed that these two distinct pathways are integrated at the transcriptional level to provide multifactorial control of cell differentiation. The activation of osteogenic genes requires the cooperation of BMPâ2 pathwayâassociated Smad1/5/8 heteromeric complexes and mechanosensitive YAP/TAZ translocation. It is further demonstrated that the Smad complexes remain bound onto and active on target genes, even after BMPâ2 removal, suggesting that they act as a âmolecular memory unit.â Thus, synergistic stimulation with BMPâ2 and mechanical cues drives osteogenic differentiation in a programmable fashion
Building a community to engineer synthetic cells and organelles from the bottom-up
Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives
The dependence of Ig class-switching on the nuclear export sequence of AID likely reflects interaction with factors additional to Crm1 exportin
Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that - beyond a requirement for threshold Crm1 binding - there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells
Designed Ankyrin Repeat Proteins as Actin Labels of Distinct Cytoskeletal Structures in Living Cells
The orchestrated assembly of actin and actin-binding
proteins into
cytoskeletal structures coordinates cell morphology changes during
migration, cytokinesis, and adaptation to external stimuli. The accurate
and unbiased visualization of the diverse actin assemblies within
cells is an ongoing challenge. We describe here the identification
and use of designed ankyrin repeat proteins (DARPins) as synthetic
actin binders. Actin-binding DARPins were identified through ribosome
display and validated biochemically. When introduced or expressed
inside living cells, fluorescently labeled DARPins accumulated at
actin filaments, validated through phalloidin colocalization on fixed
cells. Nevertheless, different DARPins displayed different actin labeling
patterns: some DARPins labeled efficiently dynamic structures, such
as filopodia, lamellipodia, and blebs, while others accumulated primarily
in stress fibers. This differential intracellular distribution correlated
with DARPinâactin binding kinetics, as measured by fluorescence
recovery after photobleaching experiments. Moreover, the rapid arrest
of actin dynamics induced by pharmacological treatment led to the
fast relocalization of DARPins. Our data support the hypothesis that
the localization of actin probes depends on the inherent dynamic movement
of the actin cytoskeleton. Compared to the widely used LifeAct probe,
one DARPin exhibited enhanced signal-to-background ratio while retaining
a similar ability to label stress fibers. In summary, we propose DARPins
as promising actin-binding proteins for labeling or manipulation in
living cells