Biology subjects

Malla, M.

Publications and source records attributed to Malla, M..

3 recordsLinked to original sources

bioRxiv2022

Cytoplasmic tail of the putative polycystin channel Pkd2 regulates its clustering in the fission yeast eisosomes

Polycystins are a family of conserved ion channels, mutations of which lead to human genetic disorder Autosomal Dominant Polycystic Kidney Disease. The unicellular model organism fission yeast Schizosacchromyces pombe possesses single essential polycystin Pkd2 that localizes to the plasma membrane and is required for cell proliferation. Here, we carried out a functional analysis of Pkd2 based on its Alphafold predicted structure. It consisted of N-terminal lipid-binding (LBD), central transmembrane (TMD) and C-terminal cytoplasmic (CCD) domains. LBD assumes a unique immunoglobulin-fold, while TMD contains nine transmembrane helices. Both were essential. Although the mostly disordered CCD was not, its removal led to clustering of Pkd2 in eisosomes, a microdomain of the plasma membrane. Inhibiting eisosome assembly prevented the clustering but disrupting ER-PM contacts further increased it. Pkd2 shared similar structure with two other putative channels Trp663 and Trp1322, but their intracellular localization and function diverged from each other. Replacing LBD with that of Trp663 partially restored the function of Pkd2, but TMD could not be replaced by either that of Trp663 or human polycystins. We concluded that both the plasma membrane microdomains and cytoplasmic tail of Pkd2 regulate the cell surface clustering of this putative ion channel.

cell biology↗

bioRxiv2022

Membrane stretching activates calcium-permeability of putative fission yeast Pkd2 channel

Pkd2 is the fission yeast homolog of polycystins. This putative ion channel localizes to the plasma membrane. It is required for the expansion of cell volume during interphase growth and cytokinesis, the last step of cell division. However, the channel activity of Pkd2 remains untested. Here, we examined the calcium permeability and mechanosensitivity of Pkd2 through in vitro reconstitution and calcium imaging of the pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayer of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypo-osmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared to the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypo-osmotic shock-triggered calcium spikes by 59%. During cytokinesis, mutations of pkd2 reduced by 60% the calcium spikes that accompany the cell separation and the ensuing membrane stretching. We concluded that fission yeast polycystin Pkd2 allows calcium influx when activated by membrane stretching, representing a likely mechanosensitive channel that contributes to the cytokinetic calcium spikes.

cell biology↗

bioRxiv2021

Counting actin in contractile rings reveals novel contributions of cofilin and type II myosins to fission yeast cytokinesis

Cytokinesis by animals, fungi and amoebas depends on actomyosin contractile rings, which are stabilized by continuous turnover of actin filaments. Remarkably little is known about the amount of polymerized actin in contractile rings, so we used low concentration of GFP-Lifeact to count total polymerized actin molecules in the contractile rings of live fission yeast cells. Contractile rings of wild-type cells accumulated polymerized actin molecules at 4,900/min to a peak number of [~]198,000 followed by a loss of actin at 5,400/min throughout ring constriction. In adf1-M3 mutant cells with cofilin that severs actin filaments poorly, contractile rings accumulated polymerized actin at twice the normal rate and eventually had almost two-fold more actin along with a proportional increase in type II myosins Myo2, Myp2 and formin Cdc12. Although 30% of adf1-M3 mutant cells failed to constrict their rings fully, the rest lost actin from the rings at the wild-type rates. Mutations of type II myosins Myo2 and Myp2 reduced contractile ring actin filaments by half and slowed the rate of actin loss from the rings.

cell biology↗