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1.
Mol Biol Cell ; 34(4): ar30, 2023 04 01.
Article in English | MEDLINE | ID: mdl-36790918

ABSTRACT

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/ß as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/ß interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/ß increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/ß have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/ß spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal-containing spindle assembly factors.


Subject(s)
Karyopherins , alpha Karyopherins , alpha Karyopherins/metabolism , Karyopherins/metabolism , Microtubules/metabolism , Kinesins/metabolism , Protein Binding/genetics , beta Karyopherins/metabolism , Microtubule-Associated Proteins/metabolism , Spindle Apparatus/metabolism
2.
Mol Biol Cell ; 32(7): 590-604, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33566676

ABSTRACT

The asymmetric distribution of microtubule (MT) dynamics in migrating cells is important for cell polarization, yet the underlying regulatory mechanisms remain underexplored. Here, we addressed this question by studying the role of the MT depolymerase, MCAK (mitotic centromere-associated kinesin), in the highly persistent migration of RPE-1 cells. MCAK knockdown leads to slowed migration and poor directional movement. Fixed and live cell imaging revealed that MCAK knockdown results in excessive membrane ruffling as well as defects in cell polarization and the maintenance of a major protrusive front. Additionally, loss of MCAK increases the lifetime of focal adhesions by decreasing their disassembly rate. These functions correlate with a spatial distribution of MCAK activity, wherein activity is higher in the trailing edge of cells compared with the leading edge. Overexpression of Rac1 has a dominant effect over MCAK activity, placing it downstream of or in a parallel pathway to MCAK function in migration. Together, our data support a model in which the polarized distribution of MCAK activity and subsequent differential regulation of MT dynamics contribute to cell polarity, centrosome positioning, and focal adhesion dynamics, which all help facilitate robust directional migration.


Subject(s)
Cell Polarity/physiology , Focal Adhesions/metabolism , Kinesins/metabolism , Cell Adhesion/physiology , Cell Movement/physiology , Centromere/metabolism , Humans , Kinesins/physiology , Microtubules/metabolism , Microtubules/physiology , Mitosis , Phosphorylation , Protein Serine-Threonine Kinases/metabolism
3.
MAbs ; 11(3): 463-476, 2019 04.
Article in English | MEDLINE | ID: mdl-30636503

ABSTRACT

Monoclonal antibodies are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (MS) to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 á´¼C). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the Fab region. At higher temperatures (e.g., 65 á´¼C), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.


Subject(s)
Diethyl Pyrocarbonate/chemistry , Immunoglobulin Fab Fragments/chemistry , Models, Molecular , Rituximab/chemistry , Mass Spectrometry , Protein Structure, Quaternary
4.
Biomolecules ; 9(1)2018 12 20.
Article in English | MEDLINE | ID: mdl-30577528

ABSTRACT

Proper regulation of microtubules (MTs) is critical for the execution of diverse cellular processes, including mitotic spindle assembly and chromosome segregation. There are a multitude of cellular factors that regulate the dynamicity of MTs and play critical roles in mitosis. Members of the Kinesin-8 family of motor proteins act as MT-destabilizing factors to control MT length in a spatially and temporally regulated manner. In this review, we focus on recent advances in our understanding of the structure and function of the Kinesin-8 motor domain, and the emerging contributions of the C-terminal tail of Kinesin-8 proteins to regulate motor activity and localization.


Subject(s)
Kinesins/metabolism , Microtubules/metabolism , Chromosome Segregation , Humans , Kinesins/chemistry , Microtubules/chemistry , Mitosis , Protein Domains , Spindle Apparatus/physiology
5.
Sci Rep ; 8(1): 5820, 2018 04 11.
Article in English | MEDLINE | ID: mdl-29643409

ABSTRACT

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.


Subject(s)
Capsid Proteins/metabolism , Parechovirus/physiology , RNA, Viral/metabolism , Virion/metabolism , Virus Assembly , Capsid Proteins/chemistry , Cross-Linking Reagents/chemistry , HT29 Cells , Humans , Models, Molecular , RNA, Viral/chemistry
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