Chandrasekar Lab

Sanford Research - Chandrasekar Lab 

Cells have compartmentalized structures and rely on regulated membrane trafficking to move cargo between organelles/compartments. Many essential cellular processes such as nutrient uptake, receptor mediated signaling and synaptic vesicle recycling depend on membrane trafficking and endocytosis. Our lab is interested in the role of actin cytoskeleton and actin associated proteins in membrane trafficking and endocytic mechanisms in cells. Recently, we demonstrated that nonmuscle myosin II (MII), an actin associated motor protein, is critical for both compensatory and constitutive receptor mediated endocytosis in neurons and fibroblasts. Our work provided the first cellular and genetic evidence for the importance of actin-MII interactions during early stages of clathrin coated vesicle formation. Our current model is that MII regulates endocytosis by forming a contractile network with actin around the site of membrane remodeling and acting like a purse string to regulate the curvature and scission processes, thereby aiding in vesicle budding (Fig.1). Identifying MII as a critical player in endocytosis has raised the following questions:
 
  1. Does actin-MII play a central role in all types of membrane trafficking events?
     
  2. What are the mechanisms by which MII participates and selectively regulates this crucial cellular process?
     
  3. What are the physiological consequences of loss of actin-MII mediated membrane trafficking in vertebrate systems?
Our work on primary neurons indicates that MII-driven tension/actin dynamics regulate the major pathway of synaptic vesicle retrieval (clathrin mediated compensatory endocytosis) and is critical for synaptic transmission. This suggests a unique and essential role for actin-MII mediated tension in cell types that require rapid and continuous membrane remodeling to perform essential organ function. We are interested in understanding the cell type specific requirements for dynamic cytoskeletal changes in cells that populate organs like kidney and brain.
 
Chandrasekar Lab - MII in CME
Fig.1: Model showing the role of MII in clathrin mediated endocytosis (CME). Actin polymerization drives the vesicle away from the plasma membrane through its interaction with the clathrin coat (mediated by Hip1R) (upward black arrow). Simultaneously, MIIB bipolar filaments contract, producing tension on the actin network associated with the coated pit, arranged parallel to the plasma membrane. The interaction of this actin with dynamin directly or through cortactin produces a purse string-like tightening of the neck and a shearing action orthogonal to the direction of vesicle movement (horizontal black arrow), facilitating scission, invagination, curvature generation and symmetry of clathrin coated pit during CME. Pre-peer reviewed version of Chandrasekar et al, Traffic. 2014 Apr;15(4):418-32.
 
To answer these questions, we employ animal and cell culture model systems. We use advanced microscopy techniques like live cell confocal imaging to monitor dynamic changes in recruitment/uptake and endosomal/lysosomal processing of fluorescently tagged ligands and receptors, clathrin coated pit markers, membrane markers, etc. We also use TIRF microscopy to analyze live surface membrane dynamics in various cell types (Fig.2).  The basic knowledge and insights obtained from our work on these fundamental cellular processes might provide mechanistic understanding of trafficking related diseases, certain renal diseases and MYH9 related disorders.
 
Chandrasekar Lab - TIRF
Fig.2: Total internal reflection fluorescence (TIRF) and Confocal images of mouse embryonic fibroblasts. Fibroblasts expressing YFP-Clathrin light chain were imaged using a Nikon A1 Ti Eclipse microscope in the Sanford Research Imaging Core. Panels show representative images of the cells in confocal (A,B) and TIRF (A’, B’) modes.
 
Contact Information for the Chandrasekar Lab:
 
Indra Chandrasekar, Ph.D.
Assistant Scientist, Sanford Children's Health Research Center
Sanford Research Center, Room 2250
2301 E. 60th Street North
Sioux Falls, SD 57104
Telephone: 605-312-6412
Fax: 605-312-6071
Email: Indra.Chandrasekar@sanfordhealth.org