Research/Projects


Heterotrimeric Kinesin-2 - Tales to Two Tails
Heterotrimeric kinesin-2 is conserved in all organisms from Chlamydomonas to Humans. It is a complex of two different motor subunits (2α & 2β) and a non-motor accessory protein, KAP. The amino acid sequences and lengths of the C-terminal tail domains of these two motor subunits are distinctly different. Initially, the KAP was proposed to bind to the tail domains of the motor subunits and act as an universal cargo adapter for kinesin-2. We showed that it interacts with the coiled-coil stalk heterodimer instead (Doodhi et al, 2009). The stalk domains appeared to form a weak heterodimer in vitro (Doodhi et al., 2012). We propose that the KAP binding may strengthen the assembly.
All kinesin-2 family members are plus-end-directed microtubule-dependent motors. It is required for the growth and maintenance of cilia and flagella. In Clamydomonas, loss of the heterotrimeric kinesin-2 motor activity blocks the transport of various flagellar components from the basal body to the tip of the flagella. Loss of this intra-flagellar transport (IFT) causes gradual resorption of the flagella. In sensory neurons, the cilia are differentiated to function-specific forms, called sensory cilia. In C. elegans, the sensory cilium was shown to form by cooperative actions of two different forms of kinesin-2 (heterotrimeric and homodimeric). We showed that the heterotrimeric kinesin-2 is necessary and sufficient for the assembly of all types of sensory cilia in Drosophila (Sarpal et al., 2003; Jana et al., 2011). A similar result was obtained in zebra fish later. We are currently studying the growth mechanisms of the bipartite olfactory cilia in the Drosophila antenna, as well as the role of odour stimulation in the odorant receptor (OR) transport into the cilia.
Previous studies in mouse and Drosophila had also shown that kinesin-2 is involved in the axonal transport. We found that it is particularly involved in the synaptic enrichment of the choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in Drosophila (Baqri et al., 2006). ChAT directly binds to the C-terminal tail domain of kinesin-2α, which is essential for its transport (Sadananda et al., 2012). We have also identified two different sets of proteins (both soluble and transmembrane) interacting with two different tails of the kinesin-2 motor subunits. Currently we are studying their biological implications.
However, the mechanisms of interactions with its cargoes are still poorly understood. It is also unclear how the two different motor subunits assemble into a single coiled-coil duplex and what is the role of accessory subunit in this complex. All these may have an important bearing on regulating a variety of the kinesin-2 based transport in the cell.

 

Spermatogenesis – From early divisions to sperm release
Sperm development and release are two essential steps regulating reproduction in multicellular organisms. Spermatogenesis, the study of sperm development, is a highly active field. We study the process in Drosophila at two distinct stages – the regulation of stem cell transit amplification and the spermiation.
Drosophila testis consists of 8-9 germline stem cells (GSCs) at the apex which are surrounded by twice the number of somatic cyst cells (CySC). Each GSC and the associated pair of CySCs divide together producing a gonialblast (GB) and two somatic cyst cells (SCC). The SCCs encapsulate the GB and the latter undergoes four mitosis producing 16-cell spermatogonial cyst. The SCCs don’t divide, but differentiates in step along with the germ cells. The interaction between SCCs and the germline plays a critical role in spermatogenesis. We showed that Dynein and Myosin V functions in the SCCs are essential for arresting the spermatogonial divisions after four rounds (Joti et al., 2011). It also regulates the expression of bag-of-marbles (bam) in the germline cells. Bam is essential for the mitotic arrest. Currently we are investigating the specific roles of the Dynein and Myosin V actions in the SCCs.

In a separate study we have discovered a new role of Dynamin in the head cyst cells (HCC) at the terminal stage in regulating the sperm release (Desai et al., 2009). We have now established an assay to observe the sperm release in live testis preparations. It is utilized to identify the underlying cellular mechanisms.