scholarly journals Profilin 1 Controls the Assembly, Organization, and Dynamics of Leading Edge Actin Structures Through Internetwork Competition and Collaboration

2019 ◽  
Author(s):  
Kristen Skruber ◽  
Peyton V. Warp ◽  
Rachael Shklyarov ◽  
James D. Thomas ◽  
Maurice S. Swanson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Leading Edge ◽  
Major Determinant ◽  
Complex Structures ◽  
Actin Assembly ◽  
Assembly Factors ◽  
Monomer Distribution ◽  
In Cells

AbstractHow actin monomers are distributed to different networks remains poorly understood. One emerging concept is that the monomer pool is limited and heterogenous, causing biased assembly and internetwork competition. However, most knowledge regarding monomer distribution comes from studies where competing networks are discrete. In metazoans, many actin-based structures are complex, containing competing networks that overlap and are functionally interdependent. Addressing how monomers control the assembly and organization of these complex structures is critical to understanding how actin functions in cells. Here, we identify the monomer-binding protein profilin 1 (PFN1) as a major determinant of actin assembly, organization, and network homeostasis in mammalian cells. At the leading edge, PFN1 controls the localization and activity of the assembly factors Arp2/3 and Mena/VASP, with discrete stages of internetwork competition and collaboration occurring at different PFN1 concentrations. This causes substantial changes to leading edge actin architecture and the types of structures that form there.

scholarly journals Physiological role of the interaction between CARMIL1 and capping protein

2013 ◽  
Vol 24 (19) ◽  
pp. 3047-3055 ◽  
Author(s):  
Marc Edwards ◽  
Yun Liang ◽  
Taekyung Kim ◽  
John A. Cooper
Keyword(s):  
Cell Migration ◽  
Mammalian Cells ◽  
Physiological Role ◽  
Physiological Significance ◽  
Actin Assembly ◽  
Capping Protein ◽  
And Control ◽  
In Cells

The regulation of free barbed ends is central to the control of dynamic actin assembly and actin-based motility in cells. Capping protein (CP) is known to regulate barbed ends and control actin assembly in cells. The CARMIL family of proteins can bind and inhibit CP in vitro, but the physiological significance of the interaction of CARMIL with CP in cells is poorly understood. Mammalian cells lacking CARMIL1 have defects in lamellipodia, macropinocytosis, cell migration, and Rac1 activation. Here we investigate the physiological significance of the CARMIL1–CP interaction, using a point mutant with a well-defined biochemical defect. We find that the CARMIL1–CP interaction is essential for the assembly of lamellipodia, the formation of ruffles, and the process of macropinocytosis. In contrast, the interaction of CARMIL1 with CP shows little to no importance for other functions of CARMIL1, including localization of CARMIL1 to the membrane, activation of Rac1, and cell migration. One implication is that lamellipodia are only marginally important for cell migration in a wound-healing model. The results also suggest that the ability of CARMIL1 to inhibit CP in cells may be regulated.

Rac1 is the small GTPase responsible for regulating the neutrophil chemotaxis compass

Blood ◽  
2004 ◽  
Vol 104 (12) ◽  
pp. 3758-3765 ◽  
Author(s):  
Chun Xiang Sun ◽  
Gregory P. Downey ◽  
Fei Zhu ◽  
Adeline L. Y. Koh ◽  
Herman Thang ◽  
...  
Keyword(s):  
Molecular Motor ◽  
Leading Edge ◽  
Cre Recombinase ◽  
Small Gtpase ◽  
Neutrophil Chemotaxis ◽  
Actin Assembly ◽  
Myeloid Lineage ◽  
Conditional Allele ◽  
In Cells

Although both of the small Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 have been demonstrated to play a role in chemotaxis, the precise and possible unique roles performed by each of these 2 Rac isoforms in neutrophil chemotaxis have not been defined. To elucidate the specific roles of Rac1 and Rac2 in neutrophils during the process of chemotaxis, we generated mice deficient in Rac1, Rac2, or in both Rac1 and Rac2 in cells of myeloid lineage including neutrophils by mating Rac2 null mice with mice carrying a conditional allele for Rac1 and expressing the Cre recombinase downstream of a specific myeloid promoter, lysozyme M. We demonstrate here that although Rac1 null neutrophils display normal chemokinesis, they are unable to migrate toward the source of the chemoattractant. By contrast, Rac2 null neutrophils can orient toward the chemoattractant source but are unable to migrate efficiently. We show that Rac1 is essential for gradient detection and orientation toward the chemoattractant source through spatially constrained regulation of phosphoinositol-3,4,5-trisphosphate (PIP3) and Akt in the leading edge and confirm that Rac2 is the primary regulator of actin assembly providing the molecular motor for neutrophil translocation during chemotaxis.

scholarly journals The kinetic landscape of an RNA-binding protein in cells

Nature ◽  
2021 ◽  
Vol 591 (7848) ◽  
pp. 152-156 ◽  
Author(s):  
Deepak Sharma ◽  
Leah L. Zagore ◽  
Matthew M. Brister ◽  
Xuan Ye ◽  
Carlos E. Crespo-Hernández ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Cells

scholarly journals SARP, a new alternatively spliced protein phosphatase 1 and DNA interacting protein

2007 ◽  
Vol 402 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Gareth J. Browne ◽  
Margarida Fardilha ◽  
Senga K. Oxenham ◽  
Wenjuan Wu ◽  
Nicholas R. Helps ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Transforming Growth Factor Β ◽  
Ankyrin Repeat ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Alternative Mrna Splicing

PP1 (protein phosphatase 1) is a ubiquitously expressed serine/threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K354VHF357) modulates endogenous SARP–PP1 activity in mammalian cells. This SARP–PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M110/MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor β inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92–95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1γ1 and PP1γ2. SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1α and PP1γ1. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.

scholarly journals Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

2011 ◽  
Vol 286 (22) ◽  
pp. 19229-19236 ◽  
Author(s):  
Laura A. Lindsey-Boltz ◽  
Aziz Sancar
Keyword(s):  
Dna Damage ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Effector Proteins ◽  
Ataxia Telangiectasia Mutated ◽  
Checkpoint Kinase ◽  
Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

scholarly journals DjA1 maintains Golgi integrity via interaction with GRASP65

2019 ◽  
Vol 30 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Jie Li ◽  
Danming Tang ◽  
Stephen C. Ireland ◽  
Yanzhuang Wang
Keyword(s):  
Heat Shock ◽  
Membrane Fusion ◽  
Structure Formation ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Golgi Membrane ◽  
Biochemical Methods ◽  
Golgi Fragmentation ◽  
Golgi Structure

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

scholarly journals Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.

1991 ◽  
Vol 115 (1) ◽  
pp. 31-43 ◽  
Author(s):  
H Plutner ◽  
A D Cox ◽  
S Pind ◽  
R Khosravi-Far ◽  
J R Bourne ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Essential Role ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Initial Step ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Golgi Compartment

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.

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