KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell

Nishaben M. Patel; Meenakshi Sundaram Aravintha Siva; Ruchi Kumari; Dipeshwari J. Shewale; Ashim Rai; Michael Ritt; Prerna Sharma; Subba Rao Gangi Setty; Sivaraj Sivaramakrishnan; Virupakshi Soppina

doi:10.1126/sciadv.abd2054

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell

Science Advances ◽

10.1126/sciadv.abd2054 ◽

2021 ◽

Vol 7 (6) ◽

pp. eabd2054

Author(s):

Nishaben M. Patel ◽

Meenakshi Sundaram Aravintha Siva ◽

Ruchi Kumari ◽

Dipeshwari J. Shewale ◽

Ashim Rai ◽

...

Keyword(s):

Single Molecule ◽

Regulatory Mechanism ◽

Coiled Coil ◽

Recycling Endosomes ◽

Single Molecule Level ◽

Cellular Processes ◽

Membrane Tubulation ◽

Guanosine Triphosphatase ◽

Neck Coil ◽

Unique Mechanism

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil–coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

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Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology ◽

10.1115/nemb2010-13039 ◽

2010 ◽

Author(s):

Kyungsuk Yum ◽

Sungsoo Na ◽

Yang Xiang ◽

Ning Wang ◽

Min-Feng Yu

Keyword(s):

Single Molecule ◽

Living Cells ◽

Endocytic Pathway ◽

Great Promise ◽

Biological Processes ◽

Temporal Precision ◽

Single Molecule Level ◽

Cellular Processes ◽

Cellular Environment ◽

Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

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Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815826116 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4275-4284 ◽

Cited By ~ 12

Author(s):

Stefan Niekamp ◽

Jongmin Sung ◽

Walter Huynh ◽

Gira Bhabha ◽

Ronald D. Vale ◽

...

Keyword(s):

Single Molecule ◽

Open Source Software ◽

Single Molecules ◽

Coiled Coil ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Distance Measurements ◽

Single Molecule Level ◽

Wide Range ◽

Different Color

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil “stalk” of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

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Structural insights into WHAMM-mediated cytoskeletal coordination during membrane remodeling

The Journal of Cell Biology ◽

10.1083/jcb.201204010 ◽

2012 ◽

Vol 199 (1) ◽

pp. 111-124 ◽

Cited By ~ 22

Author(s):

Qing-Tao Shen ◽

Peter P. Hsiue ◽

Charles V. Sindelar ◽

Matthew D. Welch ◽

Kenneth G. Campellone ◽

...

Keyword(s):

Intracellular Transport ◽

Coiled Coil ◽

Tubular Structures ◽

Terminal Portion ◽

Actin Nucleation ◽

Cellular Processes ◽

Coiled Coil Region ◽

Membrane Tubulation ◽

Underlying Mechanisms ◽

Structural Insights

The microtubule (MT) and actin cytoskeletons drive many essential cellular processes, yet fairly little is known about how their functions are coordinated. One factor that mediates important cross talk between these two systems is WHAMM, a Golgi-associated protein that utilizes MT binding and actin nucleation activities to promote membrane tubulation during intracellular transport. Using cryoelectron microscopy and other biophysical and biochemical approaches, we unveil the underlying mechanisms for how these activities are coordinated. We find that WHAMM bound to the outer surface of MT protofilaments via a novel interaction between its central coiled-coil region and tubulin heterodimers. Upon the assembly of WHAMM onto MTs, its N-terminal membrane-binding domain was exposed at the MT periphery, where it can recruit vesicles and remodel them into tubular structures. In contrast, MT binding masked the C-terminal portion of WHAMM and prevented it from promoting actin nucleation. These results give rise to a model whereby distinct MT-bound and actin-nucleating populations of WHAMM collaborate during membrane tubulation.

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Studies of bacterial topoisomerases I and III at the single-molecule level

Biochemical Society Transactions ◽

10.1042/bst20120297 ◽

2013 ◽

Vol 41 (2) ◽

pp. 571-575 ◽

Cited By ~ 14

Author(s):

Ksenia Terekhova ◽

John F. Marko ◽

Alfonso Mondragón

Keyword(s):

Single Molecule ◽

Topoisomerase I ◽

Dna Topology ◽

Biological Molecules ◽

Single Molecule Level ◽

Cellular Processes ◽

Topoisomerase Iii ◽

Type Ia ◽

Dna Strand

Topoisomerases are the enzymes responsible for maintaining the supercoiled state of DNA in the cell and also for many other DNA-topology-associated reactions. Type IA enzymes alter DNA topology by breaking one DNA strand and passing another strand or strands through the break. Although all type IA topoisomerases are related at the sequence, structure and mechanism levels, different type IA enzymes do not participate in the same cellular processes. We have studied the mechanism of DNA relaxation by Escherichia coli topoisomerases I and III using single-molecule techniques to understand their dissimilarities. Our experiments show important differences at the single-molecule level, while also recovering the results from bulk experiments. Overall, topoisomerase III relaxes DNA using fast processive runs followed by long pauses, whereas topoisomerase I relaxes DNA through slow processive runs followed by short pauses. These two properties combined give rise to the overall relaxation rate, which is higher for topoisomerase I than for topoisomerase III, as expected from many biochemical observations. The results help us to understand better the role of these two topoisomerases in the cell and also serve to illustrate the power of single-molecule experiments to uncover new functional characteristics of biological molecules.

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Investigating the Dynamics of Cellular Processes at the Single Molecule Level with Semiconductor Quantum Dots

Fundamental Biomedical Technologies - Nanoparticles in Biomedical Imaging ◽

10.1007/978-0-387-72027-2_20 ◽

2007 ◽

pp. 427-441 ◽

Cited By ~ 1

Author(s):

Maxime Dahan

Keyword(s):

Quantum Dots ◽

Single Molecule ◽

Semiconductor Quantum Dots ◽

Single Molecule Level ◽

Cellular Processes

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Live-cell single-molecule labeling and analysis of myosin motors with quantum dots

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0413 ◽

2017 ◽

Vol 28 (1) ◽

pp. 173-181 ◽

Cited By ~ 13

Author(s):

Hiroyasu Hatakeyama ◽

Yoshihito Nakahata ◽

Hirokazu Yarimizu ◽

Makoto Kanzaki

Keyword(s):

Quantum Dots ◽

Single Molecule ◽

Intracellular Trafficking ◽

Glucose Transporter ◽

Insulin Treatment ◽

Single Particle Tracking ◽

Single Molecule Level ◽

Cellular Processes ◽

Myosin Motors ◽

Glucose Transporter Glut4

Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level.

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A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides

Research ◽

10.34133/2019/1050735 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Yi-Lun Ying ◽

Jie Yang ◽

Fu-Na Meng ◽

Shuang Li ◽

Meng-Ying Li ◽

...

Keyword(s):

Phosphatase Activity ◽

Single Molecule ◽

Critical Role ◽

Label Free ◽

Peptide Substrates ◽

Regulatory Pathways ◽

Single Molecule Level ◽

Cellular Processes ◽

One Step ◽

Targeted Drug Discovery

The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T4 oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery.

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